OA20793A - Compositions and methods for detecting plasmodium species nucleic acid. - Google Patents

Abstract

Disclosed are nucleic acid oligomers, including amplification oligomers, detection probes, and capture probes, for detection of Plasmodium species nucleic acid in a sample. Also disclosed are methods of specific nucleic acid amplification and detection, including amplification and detection of target nucleic acid in real time, using the disclosed oligomers, as well as corresponding reaction mixtures and kits.

Description

COMPOSITIONS AND METHODS FOR DETECTING PLASMODIUM

SPECIES NUCLEIC ACID

CROSS-REFERENCE TO RELATED APPLICATIONS

This application daims the benefit of U.S, Provisîonal Application No. 62/782,945, fïled December 20, 2018, which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and îs hereby incorporated by reference in its entirety. Saîd ASCII Copy, créâted on October 28, 2019, is named GDS_0110PC_20191028_Seq_Listing_ST25 and is 56,087 bytes in size.

BACKGROUND

Malaria is a serions disease caused by intra-erythrocyte parasites of the genus of Plasmodium. The parasites are contracted via an infected female Anopheles mosquito bite. There are iïve species of the parasite that cause malaria in humans: P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax. According to the World Health Organization (WHO) there were 216 million cases of Malaria worldwide in 2016, and of those, 445,000 were fatal.

The United States Food and Drug Administration (U.S. FDA) has implemented strict blood screening guidelines for accepting or deferring donors who hâve travelled to malariaend emic régions. Travelers are deferred for one year after traveling to an endemic région, or three years if the donor îs a former résident of an endemic région. People who hâve been diagnosed with malaria are deferred for three years after the completion of treatment and symptom free. There are no approved tests available in the U.S. to screen blood donations for Plasmodium. This requires carefi.il screening of prospective donors via a medical questionnaire.

In endemic countries, WHO recommends testing by thick blood films or using a highly sensitive enzyme immunoassay. In non-endemic countries, WHO recommends donors are deferred for six months from the last potential exposure combined with malaria antibody testing using a highly sensitive enzyme immunoassay. Donors may be re-instated if there is no evidence of malaria] antibody. There is a risk that these methods are not effective to detect low levels of parasitemia where transmission may occur.

Transfused Transmitted Malaria (TTM) has been documented in the U.S. There hâve been 46 cases of TTM reported between 1911 and 2015 and the latest case was reported in 201 S.

There is a need for a spécifie and sensitive nucleic acid test (NAT) for detecting Plasmodium species in a s ample to reduce TTM and the number of deferred donors.

SUMMARY

In one aspect, the présent invention pro vides a method for spécifie ail y detecting Plasmodium species nucleic acid in a sample. The method generally includes (1) contacting a sample, the sample suspected of containing Plasmodium species nucleic acid, with at least two oligomers for amplifying a target région of a Plasmodium species target nucleic acid, (2) performing an in vitro nucleic acid amplification reaction, where any Plasmodium target nucleic acid présent in said sample is used as a template for generating an amplification product, and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of Plasmodium species target nucleic acid in said sample. In some embodiments, the at least two amplification oligomers comprise (a) an amplification oligomer comprising a targethybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO; 163, or (ii) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO:171, SEQ ID NO: 172, or SEQ ID NO: 173. In other embodiments, the at least two amplification oligomers comprise (a¹) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and includes the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b') an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 188 and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO: 182.

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a)(i) and (b), the target-hybridizing sequence of (a)(i) is selected from SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55. In other embodiments, the target-hybridizing sequence of (a)(i) is contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165; in some such variations, the target-hybridizing sequence of (a)(i) is selected from SEQ ID NOs:21,23-25, 32, 33, and 35.

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a)(ii) and (b), the target-hybridizing sequence of (a)(îi) includes the sequence of SEQ ID NO: 167; in some such variations, the targethybridizing sequence of (a)(ii) is selected from the SEQ ID NOs:28-31, 34, 40, 41, and 49-51. In other embodiments, the target-hybridizing sequence of (a)(ii) includes the sequence of SEQ ID NO: 168; in some such variations, the target-hybridizing sequence of (a)(ii) is selected from SEQ ID NOs:38, 39, 43, 44, and 53.

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the target-hybridizing sequence of (b) is selected from SEQ ID NOs:80-82 and 85-100. In other embodiments, the targethybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172, In some variations where the targethybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98. In some variations where the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 172, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:80, 82, 85, and 87100.

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b¹), the target-hybridizing sequence of (a’) is selected from SEQ ID NOs:37, 46, 183, and 184. In other embodiments, the targethybridizing sequence of (a¹) is contained in the sequence of SEQ ID NO: 186; in some such variations, the target-hybridizing sequence of (a’) îs SEQ ID NO: 183 or SEQ ID NO: 184. In certain embodiments where the at least two amplification oligomers comprise the amplification oligomers of (a’) and (b¹), the target-hybridizing sequence of (b¹) is selected from SEQ ID NOs:83, 84, and 182. In more particular variations, the target-hybridizing sequence of (a’) is SEQ ID NO: 183 or SEQ ID NO: 184 and the target-hybridizing sequence of (b') is SEQ ID NO:182.

In some embodiments of a method as above, the amplification oligomer of (b) or (b¹) is a promoter primer or promoter provider further comprising a promoter sequence located 5’ to the target-hybridizing sequence of (b) or (b’), respectively. A particularly suitable promoter sequence îs a T7 promoter sequence such as, e.g., SEQ ID NO:179. In spécifie variations where an amplification oligomer of (b) includes a promoter sequence, the amplification oligomer of (b) comprises a sequence selected from SEQ ID NOs:57-59 and 62-77. In spécifie variations where an amplification oligomer of (b¹) includes a promoter sequence, the amplification oligomer of (b¹) comprises a sequence selected from SEQ ID NOs:60, 61, and 181.

Particularly suitable pairs of amplification oligomer target-hybridizing sequences of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82, (B) SEQ ID NO:33 and

SEQ ID NO:82, (C) SEQ ID NO:49 and SEQ ID NO:82, (D) SEQ ID NO:21 and SEQ ID NO:89, (E) SEQ ID NO:30 and SEQ ID NO:89, (F) SEQ ID NO:33 and SEQ ID NO:89, (G) SEQ ID NO:49 and SEQ ID NO:89, (H) SEQ ID NO:21 and SEQ ID NO:92, (I) SEQ ID NO:30 and SEQ ID NO:92, (J) SEQ ID NO:21 and SEQ ID NO:94, (K) SEQ ID NO:34 and SEQ ID NO:94, (L) SEQ ID NO:53 and SEQ ID NO:94, (M) SEQ ID NO:21 and SEQ ID NO:95, (N) SEQ ID NO:34 and SEQ ID NO:95, and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence (for example, a T7 promoter sequence such as, e.g., SEQ ID NO:179) located 5’ to the target-hybridizing sequence of (b).

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification olîgomers of (a) and (b), the at least two amplification oligomers comprise first and second amplification oligomers as in (a). In some such variations, the at least two amplification oligomers comprise first and second amplification oligomers as in (a)(ii). Particularly suitable first and second amplification oligomers of (a)(ii) include a first amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 168 (e.g., the target-hybridizing sequence of SEQ ID NO:53).

In other embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii). In some such embodiments, the amplification oligomer as in (a)(i) comprises a targethybridizing sequence that is contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO:165 (e.g., the target-hybridizing sequence of SEQ ID NO:21), and the amplification oligomer as in (a)(ii) comprises a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34).

In some embodiments of a method as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification oligomers comprise first and second amplification oligomers of (b). In some such embodiments, each of the first and second amplification oligomers of (b) comprises a target-hybridizing sequence that is contained in SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO:172 (e.g., a first amplification oligomer comprising the target-hybridizing sequence of SEQ ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence of SEQ ID NO:95). In some variations, each of the first and second amplification oligomers of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b). A particularly suitable promoter sequence is a T7 promoter sequence such as, e.g., SEQ ID NO: 179. In spécifie variations where each of the first and second amplification oligomers of (b) includes a promoter sequence, the first amplification oligomer of (b) comprises the sequence of SEQ ID NO:71 and the second amplification oligomer of (b) comprises the sequence of SEQ ID NO:72.

In certain embodiments of a method for detecting Plasmodium species nucleic acid in a sample as above, the method further includes purifyîng the target nucleic acid from other components in the sample before step (1). In some such variations, the purifyîng step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilîzed probe, where the target-hybridizing sequence configured to specifically hybridize to the Plasmodium species target nucleic acid. Particularly suitable capture probe oligomer target-hybridizing sequences include sequences that are up to about 30 contiguous nucléotides in length and include a sequence selected from SEQ ID NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof. In more spécifie variations, the capture probe oligomer targethybridizing sequence is selected from SEQ ID NOs:ll-I5, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof In some embodiments, the purifyîng step comprises contacting the sample with at least two capture probe oligomers (e.g., at least two capture probe oligomers as above); in some such variations, the at least two capture probe oligomers include a first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 19 and a second capture probe oligomer comprising the target-hybridizing sequence ofSEQ ID NO:20.

In some embodiments of a method for detecting Plasmodium species nucleic acid in a sample as above, the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of Plasmodium species in the sample. In some such embodiments where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. Suitable détection probe oligomer targethybridizing sequences inciude SEQ ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO:174 or SEQ ID NO:175, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations comprisîng the sequence of SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs: 148-155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in other such variations comprisîng SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs: 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:131, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In other embodiments of a method as above where the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b¹), the détection probe oligomer target-hybridizing sequence is at least about 13 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 189 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO:190 and SEQ ID NO:191, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. In some such variations, the détection probe oligomer target-hybridizing sequence is selected from the SEQ ID NOs: 125-130 and 143, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In particular variations of a method for detecting Plasmodium species nucleic acid in a sample as above, where the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), ihe amplification oligomer target-hybridizing sequence of (a), the amplification oligomer target-hybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO:30, SEQ 1D NO:82, and SEQ ID NO: 151 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO:151 or its complément; (B) SEQ ID NO:3Û, SEQ ID NO:82, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO:157 or its complément; (C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 155 or its complément; (D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:150 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 150 or its complément; (E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 155 or its complément; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (H) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (I) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO; 152 or its complément; (I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO: 158 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 158 or its complément; (K) SEQ ID NO:49, SEQ ID NO:89, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 150 or its complément; (L) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (O) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO; 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (V) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (Y) SEQ ID NO:2l, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément.

In particular variations of a method for deteetîng Plasmodium species nucleic acid in a sample as above, where the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b¹), the amplification oligomer target-hybridizing sequence of (a'), the amplification oligomer target-hybridizing sequence of (b'), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément; (B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 127 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 127 or its complément; (C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO; 128 or its complément; (D) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 143 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 143 or its complément; (E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 129 or its complément; or (F) SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément.

In some embodîments of a method as above utilizing at least one détection probe oligomer, the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide residue member of the détection probe oligomer nucléotide sequence.

In some embodîments of a method as above utilizing at least one détection probe oligomer, the détection probe oligomer further includes a détectable label such as, for example, a fluorescent or chemiluminescent label. A particularly suîtable chemîluminescent label is a chemiluininescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer. In some embodîments comprising a detectably labeled probe oligomer, the détectable label is a fluorescent label and the détection probe oligomer further includes a non-fluorescent quencher.

In some embodîments of a method as above, the deteetîng step (3) occurs during the amplifying step (2). In some such embodîments, the method utilizes a détection probe oligomer comprising a fluorescent label and a quencher (e.g., a molecular torch, a molecular beacon, or a TaqMan détection probe).

In some embodîments of a method as above utilizing at least one détection probe oligomer, the détection probe further includes a non-target-hybridizing sequence. In partîcular variations, a détection probe oligomer comprising a non-target-hybridizing sequence is a hairpin détection probe such as, e.g., a molecular beacon or a molecular torch.

In certain embodîments, a method for detecting Plasmodium species nucleic acid in a sample as above utilizes at least two détection probe oligomers. In some such embodîments, the at least two détection probe oligomers comprise first and second détection probe oligomers, where (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (B) the second détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof. In a more spécifie variations, the first détection probe oligomer 9 comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and the second détection probe oligomer comprises a target-hybridizing sequence selected from SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In certain variations of a method for detecting Plasmodium species nucleic acid in a sample as above, the in vitro nucleic acid amplification reaction at step (2) is an isothermal amplification reaction (e.g., a transcription-mediated amplification (TMA) reaction).

In certain variations of a method for detecting Plasmodium species nucleic acid in a sample as above, the amplification reaction is a real-time amplification reaction.

In some embodiments of a method for detecting Plasmodium species nucleic acid in a sample as above, the sample is a clinical sample. In some embodiments, the sample is a blood sample such as, for example, a red blood cell sample (e.g., a lysed blood cell sample or lysed red blood cell sample).

In another aspect, the présent invention provides a combination of at least two oligomers for determîning the presence or absence of Plasmodium species in a sample. The oligomer combination generally includes at least two oligomers for amplifying a target région of Plasmodium species target nucleic acid. In some embodiments, the at least two amplification oligomers comprise (a) an amplification oligomer comprising a target-hybridizing sequence (i) that îs from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO: 163, or (Ü) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173. In other embodiments, the at least two amplification oligomers comprise (a') an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO;185 and includes the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187; and (b') an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 188 and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO: 182.

In some embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a)(î) and (b), the targethybridizing sequence of (a)(i) is selected from SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55. In other embodiments, the target-hybridizing sequence of (a)(î) is contained in the sequence of SEQ 10

ID NO: 164 and includes the sequence of SEQ ID NO: 165; in some such variations, the targethybridizing sequence of (a)(i) is selected from SEQ IDNOs:21,23-25, 32, 33, and 35.

In some embodiments of an oligomer combination as above where the ai least two amplification oligomers comprise the amplification oligomers of (a)(ii) and (b), the targethybridizing sequence of (a)(ii) includes the sequence of SEQ ID NO: 167; in some such variations, the target-hybridizing sequence of (a)(ii) is selected from the SEQ ID NOs:28-3 1, 34, 40, 41, and 49-51. In other embodiments, the target-hybridizing sequence of (a)(ii) includes the sequence of SEQ ID NO: 168; in some such variations, the target-hybridizing sequence of (a)(ii) is selected from SEQ ID NOs:38, 39, 43, 44, and 53.

In some embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the targethybridizing sequence of (b) is selected from SEQ ID NOs:80-82 and 85-100. In other embodiments, the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172. In some variations where the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98. In some variations where the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 172, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:80, 82, 85, and 87-100.

In some embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a’) and (b¹), the targethybridizing sequence of (a') is selected from SEQ ID NOs:37, 46, 183, and 184. In other embodiments, the target-hybridizing sequence of (a') is contained in the sequence of SEQ ID NO: 186; in some such variations, the target-hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184. In certain embodiments where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b'), the target-hybridizing sequence of (b¹) is selected from SEQ ID NOs:83, 84, and 182. In more particular variations, the target-hybridizing sequence of (a¹) is SEQ ID NO: 183 or SEQ ID NO: 184 and the target-hybridizing sequence of (b’)is SEQ ID NO: 182.

In some embodiments of an oligomer combination as above, the amplification oligomer of (b) or (b') is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b) or (b'), respectively. A particularly suitable promoter sequence is a T7 promoter sequence such as, e.g., SEQ ID NO: 179. In spécifie variations where an amplification oligoiner of (b) includes a promoter sequence, the amplification oligomer of (b) comprises a sequence selected from SEQ ID NOs: 57-59 and 62-77. In spécifie variations where an amplification oligomer of (b') includes a promoter sequence, the amplification oligomer of (b¹) comprises a sequence selected from SEQ ID NOs:60, 61, and 181.

Parti cul arly suitable pairs of amplification oligomer target-hybridizing sequences of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82, (B) SEQ ID NO:33 and SEQ ID NO:82, (C) SEQ ID NO:49 and SEQ ID NO:82, (D) SEQ ID NO:21 and SEQ ID NO:89, (E) SEQ ID NO:30 and SEQ ID NO:89, (F) SEQ ID NO:33 and SEQ ID NO:89, (G) SEQ ID NO:49 and SEQ ID NO;89, (H) SEQ ID NO:21 and SEQ ID NO:92, (I) SEQ ID NO:30 and SEQ ID NO:92, (J) SEQ ID NO:21 and SEQ ID NO:94, (K) SEQ ID NO:34 and SEQ ID NO:94, (L) SEQ ID NO:53 and SEQ ID NO:94, (M) SEQ ID NO:21 and SEQ ID NO:95, (N) SEQ ID NO:34 and SEQ ID NO:95, and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence (for example, a T7 promoter sequence such as, e.g., SEQ ID NO: 179) located 5’ to the target-hybridizing sequence of (b).

In some embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification oligomers comprise first and second amplification oligomers as in (a). In some such variations, the at least two amplification oligomers comprise first and second amplification oligomers as in (a)(ii). Particularly suitable first and second amplification oligomers of (a)(ii) include a first amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 168 (e.g., the target-hybridizing sequence of SEQ ID NO:53).

In other embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(îî). In some such embodiments, the amplification oligomer as in (a)(i) comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165 (e.g., the target-hybridizing sequence of SEQ ID NO:21), and the amplification oligomer as in (a)(îi) comprises a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34).

In some embodiments of an oligomer combination as above where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification oligomers comprise first and second amplification oligomers of (b). In some such embodiments, each of the first and second amplification oligomers of (b) comprises a targethybridizing sequence that is contained in SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172 (e.g., a first amplification oligomer comprising the targethybridizing sequence of SEQ ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence of SEQ ID NO:95). In some variations, each of the first and second amplification oligomers of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5’ to the target-hybridizing sequence of (b). A particularly suitable promoter sequence is a T7 promoter sequence such as, e.g., SEQ ID NO: 179. In spécifie variations where each of the first and second amplification oligomers of (b) includes a promoter sequence, the first amplification oligomer of (b) comprises the sequence of SEQ ID NO:7I and the second amplification oligomer of (b) comprises the sequence of SEQ ID NO:72.

In certain embodiments of an oligomer combination for detecting Plasmodium species nucleîc acid in a sample as above, the oligomer combination further includes at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, where the target-hybridizing sequence configured to specifically hybridîze to the Plasmodium species target nucleic acid. Particularly suitable capture probe oligomer target-hybridizing sequences include sequences that are up to about 30 contiguous nucléotides in length and include a sequence selected from SEQ ID NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof. In more spécifie variations, the capture probe oligomer target-hybridizing sequence is selected from SEQ ID NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof. In some embodiments, the oligomer combination includes at least two capture probe oligomers (e.g., at least two capture probe oligomers as above); in some such variations, the at least two capture probe oligomers include a first capture probe oligomer comprising the targethybridizing sequence of SEQ ID NO: 19 and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20.

In some embodiments of an oligomer combination for detecting Plasmodium species nucleic acid in a sample as above, the oligomer combination further includes at least one détection probe oligomer comprising a target-hybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifîable by the at least two amplification oligomers. In some such embodiments where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. Suitable détection probe oligomer target-hybridizing sequences include SEQ ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations comprising the sequence of SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs: 148-155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in other such variations comprising SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:131, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In other embodiments of an oligomer combination as above further including at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a¹) and (b¹), the détection probe oligomer target-hybridizing sequence is at least about 13 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 189 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO:190 and SEQ ID NO:I91, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. In some such variations, the détection probe oligomer target-hybridizing sequence is selected from the SEQ ID NOs: 125-130 and 143, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In particular variations of an oligomer combination as above further including at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a) and (b), the amplification oligomer target-hybridizing sequence of (a), the amplification oligomer target-hybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID N0:3Û, SEQ ID NO:82, and SEQ ID NO: 151 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 151 or its complément; (B) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 155 or its complément; (D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 150 or its complément; (E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 155 or its complément; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (G) SEQ ID NO;2l, SEQ ID NO:89, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (H) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (I) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO; 158 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 158 or its complément; (K) SEQ ID NO:49, SEQ ID NO:89, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 150 or its complément; (L) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (O) SEQ ID NQ:30, SEQ ID NO:92; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO:148 or its complément; (Q) SEQ ID NO:2I, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 148 or iis complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO:I48 or its complément; (V) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément; (X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (Y) SEQ ID N0:21, SEQ ID N0:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; (AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO;157 or its complément; (AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément.

In particular variations of an oligomer combination as above further including at least one détection probe oligomer and where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b¹), the amplification oligomer target-hybridizing sequence of (a’), the amplification oligomer target-hybridizing sequence of (b'), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément; (B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 127 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 127 or its complément; (C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 128 or its complément; (D) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO:143 or its complément, or a DNA équivalent or

DNA/RNA chimeric of SEQ ID NO: 143 or its complément; (E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 129 or its complément; or (F) SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément.

In some embodiments of an oligomer combination as above further including at least one détection probe oligomer, the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide resîdue member of the détection probe oligomer nucléotide sequence.

In some embodiments of an oligomer combination as above further including at least one détection probe oligomer, the détection probe oligomer further includes a détectable label such as, for example, a fluorescent or chemiluminescent label. A particularly suitable chemiluminescent label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer. In some embodiments comprising a detectably labeled probe oligomer, the détectable label is a fluorescent label and the détection probe oligomer further includes a non-fluorescent quencher; particularly suitable détection probe oligomers comprising a fluorescent label and a quencher including molecular torches, molecular beacons, and TaqMan détection probes.

In some embodiments of an oligomer combination as above further including at least one détection probe oiigomer, the détection probe further includes a non-target-hybridizing sequence. In particular variations, a détection probe oligomer comprising a non-targethybridizing sequence is a hairpin détection probe such as, e.g., a molecular beacon or a molecular torch.

In certain embodiments, an oligomer combination for detecting Plasmodium species nucleic acid in a sample as above includes at least two détection probe oligomers. In some such embodiments, the at least two détection probe oligomers comprise first and second détection probe oligomers, where (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (B) the second détection probe oiigomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof. In a more spécifie variation, the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 1 57 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and the second détection probe oligomer comprises a target-hybridizing sequence selected from SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerîcs thereof.

In another aspect, the present invention provides use of a combination of at least two oligomers as above for specifically amplifying Plasmodium species nucleic acid in a sample.

In another aspect, the present invention provides a détection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample. In some embodiments, the détection probe oligomer comprises a target-hybridizing sequence that is from about 13 to about 40 nucléotides in length and configured to specificaily hybridize to a target sequence contained within a Plasmodium species target région amplifîable by an oligomer combination comprising first and second Plasmodium-specific amplification oligomers, where (a) the first amplification oligomer comprises a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO: 163, or (ii) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and includes the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; and (b) the second amplification oligomer comprises a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173. In some such embodiments, the détection probe oligomer target-hybridizing sequence is (î) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. Suitable détection probe oligomer target-hybridizing sequences include SEQ ID NOs;131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations comprising the sequence of SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs;l48-155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in other such variations comprising SEQ ID

NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, the détection probe oligomer target-hybridizing sequence is seiected from SEQ ID NOs:147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof. In certain embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence seiected from SEQ ID NO: 177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof; in some such variations, the détection probe oligomer target-hybridizing sequence is seiected from SEQ ID NOs:I31, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In other embodiments of a détection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, the détection probe oligomer comprises a target-hybridizing sequence that is at least about 13 nucléotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target région amplifîable b y an oligomer combination comprising first and second Plasmodium-specific amplification oligomers, where (a) the first amplification oligomer comprises a targethybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and includes the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b) the second amplification oligomer comprises a target-hybridizing sequence that is contained in SEQ ID NO:188 and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182. In some such embodiments, the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 189 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence seiected from SEQ ID NO: 190 and SEQ ID NO: 191, including compléments, DNA équivalents, and DNA/RNA équivalents thereof. In more spécifie variations, the détection probe oligomer target-hybridizing sequence is seiected from the SEQ ID NOs:125-130 and 143, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In some embodiments of a détection probe oligomer as above, the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide residue member of the détection probe oligomer nucléotide sequence.

In some embodiments of a détection probe oligomer as above, the détection probe oligomer further includes a détectable label such as, for example, a fluorescent or chemiluminescent label. A particularly suitable chemiluminescent label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer. In some embodiments comprising a détectable label, the détectable label is a fluorescent label and the détection probe oligomer further includes a non-fluorescent quencher; particularly suitable détection probe oligomers comprising a fluorescent label and a quencher including molecular torches, molecular beacons, and TaqMan détection probes.

In some embodiments of a détection probe oligomer as above, the détection probe further includes a non-target-hybrîdizing sequence. In particular variations, a détection probe oligomer comprising a non-target-hybrîdizing sequence is a hairpin détection probe such as, e.g., a molecular beacon or a molecular torch.

In another aspect, the présent invention provides a combination of at least two oligomers for detecting a Plasmodium species target nucleic acid in a sample, the oligomer combination comprising at least two détection probe oligomers as above. In some embodiments, the at least two détection probe oligomers comprise a (A) a first détection probe oligomer comprising a target-hybrîdîzing sequence that (i) is contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ 1D NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (B) a second détection probe oligomer comprising a target-hybrîdîzing sequence that (i) is contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO: 176 or îts complément, or a DNA équivalent or DNA/RNA chimeric thereof. In more spécifie variations, the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and the second détection probe oligomer comprises a target-hybridizing sequence selected from SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

In another aspect, the présent invention provides use of a détection probe oligomer or oligomer combination according as above for specifically detecting Plasmodium species nucleic acid in a sample.

In another aspect, the présent invention provides a capture probe oligomer for specifically isolating Plasmodium species nucleic acid from a sample. In some embodiments, the capture probe oligomer comprises a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an îmmobilîzed probe, where the target-hybridizing sequence is up to about 30 contiguous nucléotides in length and includes a sequence selected from SEQ ID NOs:ll-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof. In more spécification variations, the capture probe oligomer target-hybridizing sequence is selected from SEQ ÎD NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

In another aspect, the présent invention provides a combination of at least two oligomers for specifically isolating Plasmodium species nucleic acid froiu a sample, the oligomer combination comprising at least two capture probe oligomers as above. In some embodiments, the at least two capture probe oligomers comprise a first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 19, or a DNA équivalent or DNA/RNA chimeric thereof, and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20, or a DNA équivalent or DNA/RNA chimeric thereof.

In another aspect, the présent invention provides use of a capture probe oiîgomer or oligomer combination as above for specifically capturing Plasmodium species nucleic acid from a sample.

In another aspect, the présent invention provides a kit comprising a combination of at least two oligomers as above.

In another aspect, the présent invention a reaction mixture comprising a combination of at least two oligomers as above.

These and other aspects of the invention will become évident upon reference to the following detailed description of the invention and the attached drawings.

DEFINITIONS

Unless defïned otherwise, ail technical and scîentific terrns used herein hâve the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following ternis and phrases hâve the meanings ascribed to them unless specified otherwise.

The terrns a, an, and the include plural referents, unless the context clearly indicates otherwise.

Sample includes any specimen that may contain, or is suspected of containing, Plasmodium species or components thereof, such as nucleic acids or fragments of Plasmodium nucleic acids. The sample may be an isolated sample. Samples include bîological samples which include any tissue or material derived from a living or dead human that may contain the Plasmodium parasite or components thereof (e.g., a target nucleic acid derived therefrom), including, e.g., blood, peripheral blood and red blood cells. The use of other sample types that may contain the Plasmodium parasite or components thereof (e.g., a target nucleic acid derived therefrom) - such as plasma, sérum, lymph no de, gastrointestinal tissue, faeces, urine, semen or other body fluids or materials - is also contemplated. The biological sample may be treated to physically or mechanically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, dctcrgents and the like, which are used to préparé, using standard methods, a biological sample for analysis. For example, a sample may be treated with cell lysis reagent such as, e.g., a lysis reagent as described in US Pat. No. 10,093,989 or PCT Pub. No. WO 2017/189746, each incorporated by reference herein. Also, samples may include processed samples, such as those obtained from passing samples over or through a filtering device, or following centrifugation, or by adhérence to a medium, matrix, or support.

Nucleic acid refers to a multimeric compound comprising two or more covalently bonded nucleosîdes or nucleoside analogs having nitrogenous heterocyclic bases, or base analogs, where the nucleosîdes are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof. A nucleic acid backbone may be made up of a variety of linkages, including one or more of sugar -phosphodiester linkages, peptide-nucleic acid bonds (in peptide nucleic acids or PNAs, see PCT Pub. No. WO 95/32305), phosphorethioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid may be eîther ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions and 2' halide substitutions (e.g., 2’-F). Nitrogenous bases may be conventionai bases (A, G, C, T, U), analogs thereof (e.g., inosine, 5-methylisocytosine, isoguanine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 1 Ith ed., 1992, BioTechniques (2007) 43:617-24), which include dérivatives of purine or pyrimidine bases (e.g., N4-methyl deoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituent groups at the 5 or 6 position, purine bases having an altered or replacement substituent at the 2, 6 and/or 8 position, such as 2-amino-6-inethylarninopurine, 06methyl guanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazîne-pyrimidines, and 04-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine; US Pat. Nos. 5,378,825, 6,949,367 and PCT Pub. No. WO 93/13121). Nucleic acids may include abasic residues in which the backbone does not include a nitrogenous base for one or more residues (US Pat. No. 5,585,481). A nucleic acid may comprise only conventionai sugars, bases, and linkages as found in RNA and DNA, or may include conventionai components and substitutions (e.g., conventionai bases linked by a 2' methoxy backbone, or a nucleic acid including a mixture of conventionai bases and one or more base analogs). Nucleic acids may include locked nucleic acids (LNA), in which one or more nucléotide monomers hâve a bicyclic furanose unît locked in an RNA mimicking sugar conformation, which enhances hybridization affinity tôward complementary sequences in singlestranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Biochemistry (2004) 43:13233-41). Nucleic acids may include modified bases to alter the function or behavior of the nucleic acid, e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucléotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well-known in the art.

The term polynucleotide, as used herein, dénotés a nucleic acid chain. Throughout this application, nucleic acids are designated by the 5'-terminus to the 3’-tenninus. Standard nucleic acids, e.g., DNA and RNA, are typically synthesized 5'-to-3', i.e., by the addition of nucléotides to the 3'-terminus of a growîng nucleic acid.

A nucléotide, as used herein, is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2’-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2’ position of the ribose (2’-0-Me).

A nucleic-acid-based détection assay, as used herein, is an assay for the détection of a target sequence within a target nucleic acid and utilizing one more oligonucleotides that specifically hybridize to the target sequence.

In certain embodiments, a nucleic-acid-based détection assay is an amplificationbased assay, i.e., an assay that utîlizes one or more steps for amplifying a nucleic acid target sequence. Varions amplification methods for use in détection assays are known in the art, several of which are summarized further herein. For the sake of clarity, an amplificatîon-based assay may include one or more steps that do not amplify a target sequence, such as, for example, steps used in non-amplification-based assay methods (e.g., a hybridization assay or a cleavage-based assay).

In other embodiments, a nucleic-acid-based détection assay is a non-amplificationbased assay, i.e., an assay that does not rely on any step for amplifying a nucleic acid target sequence. For the sake of clarity, a nucleic-acid-based détection assay that includes a reaction for extension of a primer in the absence of any corresponding downstream amplification oligomer (e.g., extension of a primer by a reverse transcriptase to generate an RNA:DNA duplex followed by an RNase digestion of the RNA, resulting in a single-stranded cDNA complementary to an RNA target but without generating copies of the cDNA) is understood to be a non-amplificationbased assay.

An exemplary non-amplification-basée! assay is a cleavage-based assay, which is an assay that relies on the spécifie cleavage, by a flap endonuclease, of a linear duplex cleavage structure formed b y the spécifie hybridization of overlapping oligonucleotides to a target nucleic acid. In these assays, a probe oligonucleotide containîng a non-target-hybridîzing flap région is cleaved in an overlap-dependent manner by the flap endonuclease to release a cleavage product that is then detected. The principles of cleavage-based assays are well-known in the art, and exemplary assays are described in, for example, Nat. Biotechnol. (1999) 17:292-296, Mol. Diagn. (1999) 4: 135-144, J. Clin. Microbiol. (2006) 44:3443-3447, and US Patent Nos. 5,846,717, 6,706,471 and 5,614,402. Cleavage-based assays include, e.g., the commercîally availabié Invader® assays (Hologic, Inc., Madison, WI).

A target nucleic acid, as used herein, is a nucleic acid comprising a target sequence to be detected. Target nucleic acids may be DNA or RNA as described herein, and may be either single-stranded or double-stranded. The target nucleic acid may include other sequences besides the target sequence.

By isolated it îs meant that a sample containîng a target nucleic acid is taken from its natural milieu, but the term does not connote any degree of purification.

The term target sequence, as used herein, refers to the partîcular nucléotide sequence of a target nucleic acid that is to be detected. The target sequence includes the complexing sequences to which oligonucleotides (e.g., probe oligonucleotide, priming oligonucleotides and/or promoter oligonucleotides) complex during a détection process (e.g., an amplification-based détection assay such as, for example, TMA or PCR, or a non-amplificationbased détection assay such as, for example, a cleavage-based assay). Where the target nucleic acid is originally single-stranded, the term target sequence will also refer to the sequence complementary to the target sequence as présent in the target nucleic acid. Where the target nucleic acid is originally double-stranded, the term target sequence refers to both the sense (+) and antisense (-) strands. In choosing a target sequence, the skilled artisan will understand that a unique sequence should be chosen so as to distinguish between unrelated or closely related target nucleic acids.

Target-hybridizing sequence is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence. Preferably, the targethybridizing sequences are configured to specifically hybridize with a target nucleic acid sequence. Target-hybridizing sequences may be 100% complementary to the portion of the target sequence to which they are configured to hybridize, but not necessarily. Target-hybridizing sequences may also înclude inserted, deleted and/or substituted nucléotide residues relative to a target sequence. Less than 100% complementarity of a target-hybridizing sequence to a target sequence may arise, for example, when the target nucleic acid is a plurality strains within a species, such as would be the case for an oligomer configured to hybridize to the varions strains of Plasmodium. It is understood that other reasons exist for configuring a target-hybridizing sequence to hâve less than 100% complementarity to a target nucleic acid.

The tenu targets a sequence, as used herein in référencé to a région of Plasmodium sp. nucleic acid, refers to a process whereby an oligonucleotide hybridizes to the target sequence in a manner that allows for détection as described herein. In one embodiment, the oligonucleotide is complementary with the targeted Plasmodium sp. nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted Plasmodium sp. nucleic acid sequence. Preferably, the oligonucleotide that hybridizes to the target nucleic acid sequence includes at least 10 to as many as 50 nucléotides complementary to the target sequence. It is understood that at least 10 and as many as 50 is an inclusive range such that 10, 50 and each whole number there between are included. Preferably, the oligomer specifically hybridizes to the target sequence.

The term configured to dénotés an actual arrangement of the polynucleotide sequence configuration of a referenced oligonucleotide target-hybridizing sequence. For example, oligonucleotides that are configured to specifically hybridize to a target sequence hâve a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

The term configured to specifically hybridize to as used herein means that the target-hybridizing région of an oligonucleotide is designed to bave a polynucleotide sequence that could target a sequence of the referenced Plasmodium sp. target région. Such an oligonucleotide is not limited to targeting that sequence only, but is rather useful as a composition, in a kit or in a method for targeting a Plasmodium sp. target nucleic acid. The oligonucleotide is designed to fonction as a component of an assay for détection of Plasmodium sp. from a sample, and therefore is designed to target Plasmodium sp. in the presence of other nucleic acids commonly found in testing samples. Specifically hybridize to does not mean exclusively hybridize to, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, specifically hybridize to means that the oligonucleotide is configured to fonction in an assay to primarily hybridize the target so that an accurate détection of target nucleic acid in a sample can be determined. The term configured to dénotés an actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target-hybridizing sequence.

The term fragment,” as used herein in reference to a Plasmodium sp. targeted nucleic acid, refers to a piece of contiguous nucleic acid.

The term région, as used herein, refers to a portion of a nucleic acid wherein the portion is smaller than the entire nucleic acid. For example, when the nucleic acid in reference is an oligonucleotide promoter primer, the term région may be used refer to the smaller promoter portion of the entire oligonucleotide. As a non-lîmiting example, when the nucleic acid in reference is an amplicon, the term région may be used to refer to the smaller nucléotide sequence identified for hybridization by the target-hybridizing sequence of a probe.

The interchangeable ternis oligomer, oligo, and oligonucleotide” refer to a nucleic acid having generally less than 1,000 nucléotide (nt) residues, including polymers in a range having a lower limit of about 5 nt residues and an upper limit of about 500 to 900 nt residues. In some embodiments, oligonucleotides are in a size range having a lower limit of about 12 to 15 nt and an upper limit of about 50 to 600 nt, and other embodiments are in a range having a lower limit of about 15 to 20 nt and an upper limit of about 22 to 100 nt. Oligonucleotides may be purified from naturally occurring sources or may be synthesized using any of a varîety of well-known enzymatic or Chemical methods. The term oligonucleotide does not dénoté any particular fonction to the reagent; rather, it is used generically to cover ali such reagents described herein. An oligonucleotide may serve varions different fonctions. For example, it may fonction as a primer if it is spécifie for and capable of hybridizing to a complementary strand and can further be extended in the presence of a nucleic acid polymerase; it may fonction as a primer and provide a promoter if it contaîns a sequence recognized by an RNA polymerase and allows for transcription (e.g., a T7 Primer); and it may fonction to detect a target nucleic acid if it is capable of hybridizing to the target nucleic acid, or an amplicon thereof, and further provides a detectible moiety (e.g., an acridinium-ester compound).

As used herein, an oligonucleotide can substantially correspond to a specified reference nucleic acid sequence, which means that the oligonucleotide is sufficîently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence in that it would hybridize with the same target nucleic acid sequence under stringent hybridization conditions. One skilled in the art will understand that substantially corresponding oligonucleotides can vary from a reference sequence and still hybridize to the same target nucleic acid sequence. It is also understood that a first nucleic acid corresponding to a second nucleic acid includes the RNA and DNA thereof and includes the compléments thereof, unless the context clearly dictâtes otherwise. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide substantîally corresponds to a référencé nucleic acid sequence if these percentages of base identity or complementarity are from 100% to about 80%. In preferred embodiments, the percentage is from 100% to about 85%. In more preferred embodiments, this percentage is from 100% to about 90%; in other preferred embodiments, this percentage is from 100% to about 95%. Similarly, a région of a nucleic acid or amplified nucleic acid can be referred to herein as corresponding to a référencé nucleic acid sequence. One skilled in the art will understand the various modifications to the hybridization conditions that might be required at various percentages of complementarity to allow hybridization to a spécifie target sequence without causing an unacceptable level of non-specific hybridization.

Exemplary sequences for Plasmodium sp. target nucleic acid are shown in Table 19, infra. Specifically, SEQ ID NOs:180 and 192-195 are référencé sequences corresponding to rîbosomal RNA sequences for Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi. Plasmodium ovale, and Plasmodium malariae, respectively. Where a target région of Plasmodium sp. is described herein as corresponding to a defined région of SEQ ID NO: 180, it is understood that such référencé includes homologous régions of any one or more of SEQ ID NOs: 192-195. It is also understood that such reference to a région corresponding to a defined région of SEQ ID NO: 180 includes homologous régions of naturally occurring variants of any one or more of SEQ ID NOs: 180 and 192-195 that may be présent in a sample.

An amplification oligomer is an oligomer at least the 3'-end of which is complementary to a target nucleic acid and which hybridîzes to a target nucleic acid, or its complément, and participâtes in a nucleic acid amplification reaction. An example of an amplification oligomer is a primer that hybridîzes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase in an amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3' blocked end) but participâtes in or facilitâtes amplification. For example, the 5’ région of an amplification oligonucleotide may include a promoter sequence that is non-complementary to the target nucleic acid (which may be referred to as a promoter primer or promoter provider). Those skilled in the art will understand that an amplification oligomer that functions as a primer may be modified to include a 5' promoter sequence, and thus function as a promoter primer. Incorporating a 3' blocked end further modifies the promoter primer, which is now capable of hybridizing to a target nucleic acid and providing an upstream promoter sequence that serves to initiate transcription, but does not provide a primer for oligo extension. Such a modified olîgo is referred to herein as a promoter provider oligomer. Size ranges for amplification oligonucleotides include those that are about 10 to about 70 ni long (not including any promoter sequenee or poly-A taîls) and contain at least about 10 contiguous bases, or even at least 12 contiguous bases that are complementary to a région of the target nucleic acid sequenee (or a complementary strand thereof), The contiguous bases are at least 80%, or at least 90%, or completely complementary to the target sequenee to which the amplification oligomer binds. An amplification oligomer may optionally include modified nucléotides or analogs, or additîonal nucléotides that participate in an amplification reaction but are not complementary to or contained in the target nucleic acid, or template sequenee. It is understood that when referring to ranges for the length of an oligonucleotide, amplicon, or other nucleic acid, that the range is inclusive of ali whole numbers (e.g., 19-25 contiguous nucléotides in length includes 19, 20, 21, 22, 23, 24 & 25).

As used herein, a promoter is a spécifie nucleic acid sequenee that is recognized by a DNA-dependent RNA polymerase (transcriptase) as a signal to bind to the nucleic acid and begin the transcription of RNA at a spécifie site.

As used herein, a promoter provider or provider refers to an oligonucleotide comprising first and second régions and which is modified to prevent the initiation of DNA synthesis from its 3' -terminus. The first région of a promoter provider oligonucleotide comprises a base sequenee which hybridizes to a DNA template, where the hybridizing sequenee is situated 3', but not necessarily adjacent to, a promoter région. The hybridizing portion of a promoter oligonucleotide is typically at least 10 nucléotides in length and may extend up to 50 or more nucléotides in length. The second région comprises a promoter sequenee for an RNA polymerase. A promoter oligonucleotide is engineered so that it is incapable of being extended by an RNA- or DNA-dependent DNA polymerase, e.g., reverse transcriptase, preferably comprising a blocking moiety at its 3'-tenninus as described above. As referred to herein, a T7 Provider is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequenee that is recognized by T7 RNA polymerase.

Amplification'’ refers to any known procedure for obtaining multiple copies of a target nucleic acid sequenee or its complément or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothennal amplification methods. In some embodiments, isothennal amplification methods are preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase-mediated amplification uses self-replicating RNA molécules, and a replicase such as QB-replicase (e.g., US Pat. No. 4,786,600). PCR amplification uses a DNA polymerase, pairs of primers, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or from a cDNA (e.g., US Pat. Nos. 4,683,195, 4,683,202, and 4,800, 159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and dénaturation (e.g., US Pat. No. 5,427,930 and US Pat. No. 5,516,663). SD A uses a primer that contains a récognition site for a restriction endonuclease and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes the target sequence, whereby amplification occurs in a sériés of primer extension and strand displacement steps (e.g, US Pat. No. 5,422,252; US Pat. No. 5,547,861; and US Pat. No. 5,648,211). Preferred embodiments use an amplification method suitable for the amplification of RNA target nucleic acids, such as transcription-mediated amplification (TMA) or NASBA, but it will be apparent to persons of ordînary skill in the art that oligomers disclosed herein may be readily used as primers in other amplification methods.

Transcription-associated amplification, also referred to herein as transcriptionmediated amplification (TMA), refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template complementary oligonucleotide that includes a promoter sequence, and optionally may inciude one or more other oligonucleotides. TMA methods are embodiments of amplification methods used for amplifying and detecting Plasmodium target sequences as described herein. Variations of transcription-associated amplification are well-known in the art as previously disclosed in detail (e.g., US Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302, WO 88/10315, and WO 95/03430). The person of ordinary skill in the art will appreciate that the disclosed compositions may be used in amplification methods based on extension of oligomer sequences by a polymerase.

As used herein, the term real-tîme TMA refers to transcription-mediated amplification (TMA) of target nucleic acid that is monitored by real-time détection means.

The term amplicon, which is used interchangeably with amplification product, refers to the nucleic acid molécule generated during an amplification procedure that is complementary or homologous to a sequence contained within the target sequence. These terms can be used to refer to a single strand amplification product, a double strand amplification product, or one of the strands of a double strand amplification product.

Probe, détection probe, détection oligonucleotide, and détection probe oligomer are used interchangeably herein to refer to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow détection of the target sequence or amplified nucleic acid. Détection may either be direct (e.g., a probe hybridized directly to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). Probes may be DNA, RNA, analogs thereof, or combinations thereof and they may be labeled or unlabeled. A probe's target sequence generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that hybridizes specifically to at least a portion of a probe oligomer by standard base pairing. A probe may comprise target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., US Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. No. 20060068417). In a preferred embodiment, the détection probe comprises a 2' methoxy backbone, which can resuit in a higher signal being obtained.

The term TaqMan® probe refers to détection oligonucleotides that contain a fluorescent dye, typically on the 5' base, and a non-fluorescent quenching dye (quencher), typically on the 3' base. When irradiaied, the excited fluorescent dye transfers energy to the nearby quenching dye molécule rather than fluorescîng, resulting in a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe to separate the fluorophore from the quencher, thereby allowing an unquenched signal to be emitted from the fluorophore as an indicator of amplification.

As used herein, a label refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a détectable signal. Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions, e.g., hydrogen bonds, hydrophobie and ionic interactions, or formation of chelates or coordination complexes. Indirect labeling can occur through use of a bridging moiety or linker such as a binding pair member, an antibody or additional oligomer, which is either directly or indirectly labeled, and which may amplify the détectable signal. Labels include any détectable moiety, such as a radionuclide, ligand (e.g., biotîn, avidin), enzyme or enzyme substrate, reactive group, or chromophore (e.g., dye, particle, or bead that imparts détectable color), luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophore. Labels may be détectable in a homogeneous assay in which bound labeled probe in a mixture exhibits a détectable change different from that of an unbound labeled probe, e.g., instabilîty or differential dégradation properties. A homogeneous détectable label can be detected without physically removing bound from unbound forms of the label or labeled probe (e.g., US Pat. Nos. 5,283, 174, 5,656,207, and 5,658,737). Labels include chemi luminescent compounds, e.g., acridinium ester (AE) compounds thaï include standard AE and dérivatives (e.g., US Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Synthesis and methods of attaching labels to nucleic acids and detecting labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; US Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). More than one label, and more than one type of label, may be présent on a particular probe, or détection may use a mixture of probes in which each probe is labeled with a compound that produces a détectable signal (e.g., US Pat. Nos. 6, 180,340 and 6,350,579).

As used herein, structures referred to as molecular torches are designed to include distinct régions of self-complementarîty (the closing domain) which are connected by a joining région (the target-binding domain) and which hybridize to one another under predetermined hybridization assay conditions. Ail or part of the nucléotide sequences comprising closing domains may also fonction as target-binding domains. Thus, closing domains can include targetbinding sequences, non-target binding sequences, and combinations thereof.

As used herein, structures referred to as molecular beacons are designed to include a target-binding sequence flanked on both its 5' and 3' ends by sequences that are complementary to each other and which hybridize to each other under predetermined hybridization assay conditions. The flanking, complementary régions may be referred to as switch sequences.

Capture probe, capture oligonucleotide, target capture oligonucleotide, and capture probe oligomer are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base paîring and joins to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer includes an oligonucleotide comprising two binding régions: a target hybridizing sequence and an immobilized probe-binding région. A variation of this example, the two régions may be présent on two different oligomers joined together by one or more linkers. In another embodiment of a capture oligomer, the target-hybridizing sequence is a sequence that includes random or non-random poly-GU, poly-GT, or poly U sequences to bind non-specifically to a target nucleic acid and link it to an immobilized probe on a support (see, e.g., PCT Pub. No. WO 2008/016988). The immobilized probe-binding région can be a nucleic acid sequence, referred to as a tail. Tails include a substantially homopolymeric tail of about 10 to 40 nucléotides (e.g., A10 to A40), or of about 17 to 33 nt (e.g., T3A14 to T3A3O), that bind to a complementary immobilized sequence attached to the support particle or support matrix. Thus, a non-limiting example of preferred nucleic acid tails can in some embodiments include To^Aiq. 40 sequences. Another example of a capture oligomer comprises two régions, a target-hybridizing sequence and a binding pair member that is not a nucleic acid sequence.

As used herein, an immobilized oligonucleotide, immobilized probe, or immobilized nucleic acid refers to a nucleic acid binding partner that joins a capture oligomer to a support, directly or indirectly. An immobilized probe joined to a support facilitâtes séparation of a capture probe bound target from unbound material in a sample. One embodiment of an immobilized probe is an oligomer joined to a support that facilitâtes séparation of bound target sequence from unbound material in a sample. Supports may include known materials, such as matrices and parti cl es free in solution, which may be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrène, silane, polypropylene, métal, or other compositions, of which one embodiment is magnetically attractable partiel es. Supports may be monodisperse magnetic spheres (e.g., uniform size + 5%), to which an immobilized probe is joined directly (via covalent linkage, chélation, or ionic interaction), or indirectly (via one or more linkers), where the linkage or interaction between the probe and support is stable during hybridization conditions.

DESCRIPTION

The présent invention is generally directed to methods and compositions for determining the presence or absence of the protozoan parasite Plasmodium sp. in a sample, such as, e.g., a blood sample. Suitably, the methods and compositions described herein are able to detect the presence or absence of Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and/or Plasmodium vivax. In some embodiments, the présent invention provides methods for the détection of Plasmodium sp. in a sample, where the method includes performing ampli fication-based détection of a target nucleic from Plasmodium sp. The présent invention further provides compositions (including reaction mixtures) and kits comprising a combination of oligomers for detecting Plasmodium sp. - including Plasmodium falciparum and/or Plasmodium knowlesi and/or Plasmodium malariae and/or Plasmodium ovale and/or Plasmodium vivax - in a sample. The oligomer combination generally includes at least two amplification oligomers for detecting Plasmodium sp. - including Plasmodium falciparum and/or Plasmodium knowlesi and/or Plasmodium malariae and/or Plasmodium ovale and/or Plasmodium vivax - in a sample, and may further include one or more additional oligomers as described herein for performing amplification-based détection of Plasmodium sp. - including

Plasmodium falciparum and/or Plasmodium knowlesi and/or Plasmodium malariae and/or Plasmodium ovale and/or Plasmodium vivax - such as, for example, a capture probe and/or a détection probe.

Methods for detecting the presence or absence of Plasmodium sp. in a sample from a subject general!y include perfonning a nucleic-acid-based détection assay for the spécifie détection in the sample of Plasmodium sp. nucleic acid. Nucleic-acid-based détection assays generally utilize oligonucleotides that specifically hybrîdize to a target nucleic acid of Plasmodium sp. with minimal cross-reactivity to other nucleic acids suspected of being in a sample. In some variations, an oligonucleotide or combination of oligonucleotides for nucleicacid-based détection of Plasmodium sp. has minimal cross-reactivity to Babesia sp. (e.g., B. microti) nucleic acids.

In certain aspects of the invention, a combination of at least two oligomers is provided for determining the presence or absence of Plasmodium species in a sample. Typîcally, the oligomer combination includes at least first and second amplification oligomers for amplifying a Plasmodium sp. target région corresponding to a région of SEQ ID NO: 180. In such embodiments, at least one amplification oiigomer comprises a target-hybridizing sequence in the sense orientation (sense THS) and at least one amplification oligomer comprises a targethybridizing sequence in the antîsense orientation (antisense THS), where the sense THS and antisense THS of the amplification oligomers are each configured to specifically hybridize to a Plasmodium sp. target sequence corresponding to a sequence contained within SEQ ID NO: 180, and where the target-hybridizing sequences are selected such that the Plasmodium sequence targeted by the antisense THS is situated downstream of the Plasmodium sequence targeted by the sense THS (i.e., the at least two amplification oligomers are situated such that they flank the target région to be amplified).

In some embodiments, the Plasmodium sp. target région corresponds to a région of SEQ ID NO: 180 from about nucléotide position 844 or about nucléotide position 910 to about nucléotide position 1038, about nucléotide position 1051, about nucléotide position 1060, or about nucléotide position 1077. In other embodiments, the Plasmodium sp. target région corresponds to a région of SEQ ID NO:180 from about nucléotide position 1153, about nucléotide position 1169, or about nucléotide position 1182 to about nucléotide position 1327, about nucléotide position 1354, or about nucléotide position 1382.

In some embodiments, a composition includes an amplification oiigomer comprising a Plasmodium-specïüc target-hybridizing sequence substantially corresponding to, or identical to, the sequence shown in any one of SEQ ID NOs:21-56, 80-102, and 182-184. In such variations, the oligomer combination includes at least one amplification oligomer comprising a Plasmodium-specific target-hybridizing sequence of the opposite polarity (sense vs. antisense or vice versa) as the target-hybridizing sequence of the oligomer as above, such that at least two amplification oligomers flank a target région to be amplîfied.

In some embodiments, a composition includes ( 1 ) at least one amplification oligomer comprising a Plasmodium-specific target-hybridizing région substantially correspondmg to at least one sense oligomer sequence depicted in Table 1 below, and (2) at least one amplification oligomer comprising a Plasmodium-specific target hybridizing région substantially corresponding to at least one antisense oligomer sequence depicted in Table 1. In particular 10 variations, the sense and/or antisense target-hybridizing sequence(s) of an amplification oligomer combination comprises or consists of the sense and/or antisense sequence(s) selected from Table 1.

Table L Exemplary Sense and Antisense Amplification Oligomer Target-hybridizing Sequences 15 for Amplification of Plasmodium species Target Régions

SEQ ID NO	Sequence (5’—* 3’)	Sense/Antisense1
21	AATACTACAGCATGG	Sense
22	GGAAGGCAGCAGGCGCGTA	Sense
23	AATACTACAGCATGGA	Sense
24	AATACTACAGCATGGAA	Sense
25	ATACTACAGCATGGAATA	Sense
26	AT T CAGAT GT CAGAGGT GA	Sense
27	GTATTCAGATGTCAGAGGTGA	Sense
28	GTTACGATTAATAGGAGT	Sense
29	GTTACGATTAATAGGAGTA	Sense
30	GTTACGATTAATAGGAGTAG	Sense
31	GTTACGATTAATAGGAGTAGC	Sense
32	AATACTACAGCATGGAAT	Sense
33	AATACTACAGCATGGAATA	Sense
34	TACGATTAATAGGAGT	Sense
35	TACTACAGCATGGAATA	Sense
36	TATTCAGATGTCAGAGGTGA	Sense
37	TCAGTNCCTTATGAGAAATC	Sense
38	TGGCTTAGTTACGATT	Sense
39	TGGCTTAGTTACGATTAATAG	Sense
40	TTAATAGGAGTAGCTTGGGG	Sense
41	TTACGATTAATAGGAGT	Sense
42	T T CAGAT GT CAGAG GT GA	Sense
43	TTGGCTTAGTTACGAT	Sense
44	TTGGCTTAGTTACGATTA	Sense
45	TTGGGGACATTCGTATTCAGA	Sense
46	TTTAGATTGCTTCCTTCAGT	Sense

47	TTTGAATACTANAGCA	Sense
48	ACATTCGTATTCAGATGTCAG	Sense
49	CTTAGTTACGATTAATAGGA	Sense
50	CGATTAATAGGAGTAGCTTGG	Sense
51	CTTAGTTACGATTAATAGGAGTAG	Sense
52	CTTGAATACTNCAGCA	Sense
53	GGCTTAGTTACGATTA	Sense
54	AATACTANAGCATGG	Sense
55	AATACTANAGCATGGAATA	Sense
56	AATTCTAAAGAAGAGAG	Sense
80	TTCACTCCCTTAACTTTCGTTCTTG	Antisense
81	CTTGATTAATGGAAGTATTTTAGA	Antisense
82	CTTAACTTTCGTTCTTGATTAATGGAAGT	Antisense
83	CCTACTCTTGTCTTAAACTA	Antisense
84	AAACGGCCATGCATCACCATCCAAGA	Antisense
85	CTCCCTTAACTTTCGTTCTTGATTAATGGAAGT	Antisense
86	CGACGGTATCTGATCGTCTTCACTCCC	Antisense
87	CTTAACTTTCGTTCTTGATTAATGGAAG	Antisense
88	CTTAACTTTCGTTCTTGATTAATGGAAGTA	Antisense
89	CACTCCCTTAACTTTCGTTCTTGATTAATG	Antisense
90	CACTCCCTTAACTTTCGTTCTTGATTAATGG	Antisense
91	CTTCACTCCCTTAACTTTCGTTCTTGATT	Antisense
92	CTTCACTCCCTTAACTTTCGTTCTTGAT	Antisense
93	ATCGTCTTCACTCCCTTAACTTTCGTTC	Antisense
94	CTCCCTTAACTTTCGTTCTTGATTAATG	Antisense
95	TCACTCCCTTAACTTTCGTTCTTGAT	Antisense
96	CCCTTAACTTTCGTTCTTGATTAATG	Antisense
97	CTTAACTTTCGTTCTTGATTAATG	Antisense
98	TAACTTTCGTTCTTGATTAATG	Antisense
99	ACTCCCTTAACTTTCGTTCTTGAT	Antisense
100	TCCCTTAACTTTCGTTCTTGAT	Antisense
101	AGGCAAATGCTTTCGCAGTTGTTNGTCT	Antisense
102	AGGCAAATGCTTTCGCAGTTGTTTGTCT	Antisense
182	TCAAGAAAGAGCTATNAATCTGTCAATCC	Antisense
183	GAAATCAAAGTCTTTGGGTTCTG	Sense
184	CAAAGTCTTTGGGTTCTGG	Sense

'The Sense/Antisense désignation of these sequences is for exemplary purposes only. Such désignation does not necessarîly limit a sequence to the accompanying désignation.

In some embodiments, an oligomer combination comprises (a) an amplification 5 oligomer comprising a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO: 163, or (ii) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ TD NO: 168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO:171, SEQ ID NO: 172, or SEQ ID NO: 173. In some such embodiments, the oligomer combination comprises an amplification oligomer of (a)(î) where the target-hybridizing sequence of is selected from SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55, or where the target-hybridizing sequence is contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165 (e.g., a target-hybridizing sequence selected from SEQ ID NOs:21, 23-25, 32, 33, and 35). In yet other embodiments, the oligomer combination comprises an amplification oligomer of (a)(ii) where the target-hybridizing sequence of includes the sequence of SEQ ID NO: 167 (e.g., a target-hybridizing sequence selected from the SEQ ID NOs:28-31, 34, 40, 41, and 49-51), or where the target-hybridizing sequence includes the sequence of SEQ ID NO:168 (e.g., a target-hybridizing sequence selected from SEQ ID NOs:38, 39, 43, 44, and 53). In certain embodiments of an oligomer combination as above, the targethybridizing sequence of (b) is selected from SEQ ID NOs:80-82 and 85-100. In other embodiments, the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 (e.g., a target-hybridizing sequence selected from SEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98), or is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 172 (e.g., a target-hybridizing sequence selected from SEQ ID NOs:80, 82, 85, and 87-100).

In some embodiments, an oligomer combination comprises (a¹) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and includes the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b') an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 188 and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO; 182, In some embodiments, the amplification oligomer of (a') comprises a targethybridizing sequence selected from SEQ ID NOs:37, 46, 183, and 184, or a target-hybridizing sequence contained in the sequence of SEQ ID NO:186 (e.g., a target-hybridizing sequence of SEQ ID NO: 183 or SEQ ID NO: 184). In certain embodiments, the amplification oligomers of (b') comprises a target-hybridizing sequence selected from SEQ ID NOs:83, 84, and 182.

In certain embodiments, an amplification oligomer as described herein is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the targethybridizing sequence and which is non-complementary to the Plasmodium sp. targei nucleic acid. For ex ample, in some embodiments of an oligomer combination as described herein, an amplification oligomer of (b) or (b') as described above îs a promoter primer further comprising a 5’ promoter sequence. In particular embodiments, the promoter sequence is a T7 RNA polymerase promoter sequence such as, for example, a T7 promoter sequence having the sequence shown in SEQ ID NO: 179. In spécifie variations, an amplification oligomer is a promoter primer having the sequence shown selected from SEQ ID NOs:57-77 and 181.

Table 2 shows particularly suitable combinations of amplification oligomer target5 hybridizing sequences (Amp 1 and Amp 2) for détection of Plasmodium species target nucleic acid.

Table 2. Exemplary Combinations of Amplification Oligomer Target-hybridizing Sequences.

Amp 1 (SEQ ID NO)	Amp 2 (SEQ ID NO)
30	5
33	8
49	11
21	14
30	17
33	20
49	23
21	26
30	29
21	32
34	35
53	38
21	41
34	44
53	46
183	182
184	182

In some embodiments, an oligomer combination as above includes at least two sense amplification oligomers and/or at least two antisense amplification oligomers flanking a Plasmodium sp. target région. For example, an oligomer combination may include (a) at least two amplification oligomer (e.g., two or three amplification oligomers) each comprising a targethybridizing sequence that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 16 and/or (b) at least two amplification oligomers (e.g., two or three amplification oligomers) each comprising a target-hybridizing sequence thaï is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID

NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173; in some such variations, the oligomer combination includes (a) a first amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 168 (e.g., the target-hybridizing sequence of SEQ ID NO:53). In other embodiments comprising at least two sense amplification oligomers and/or at least two antisense amplification oligomers, an oligomer combination comprises (a)(i) an amplification oligomer comprising a target-hybridizing sequence that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO:163, and (ii) an amplification oligomer comprising a target-hybridizing sequence that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO:166, and includes SEQ ID NO:167 or SEQ ID NO: 168; and/or (b) at least two amplification oligomers (e.g., two or three amplification oiigomers) each comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173; in some such variations, the oligomer combination includes (a)(i) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165 (e.g., the target-hybridizing sequence of SEQ ID NO:2i), and (a)(ii) an amplification comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167 (e.g., the target-hybridizing sequence of SEQ ID NO:34). In some embodiments of an oligomer combination as above comprising at least two amplification oligomers of (b), oligomer combination comprise first and second amplification oligomers of (b), each comprising a targethybridizing sequence that is contained in SEQ ID NO: 170 and includes the sequence of SEQ ID NO:17I or SEQ ID NO: 172 (e.g., a first amplification oligomer comprising the targethybridizing sequence of SEQ ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence of SEQ ID NO:95).

In some embodiments, an oligomer combination as described herein further comprises at least one capture probe oligomer comprising a target-hybridizing sequence configured to specîfically hybridize to Plasmodium species target nucleic acid. In some such embodiments, the capture probe oligomer comprises a target-hybridizing sequence a sequence substantially corresponding to to a sequence contained in the complément of SEQ ID NO: 180. In some embodiments, a capture probe oligomer target-hybridizing sequence ts covalently attached to a sequence or moiety that binds to an immobilized probe. Suitable capture probe oligomer targethybridizing sequences include sequences that are up to about 30 contiguous nucléotides in length and include a sequence substantially corresponding to a sequence selected from SEQ ID NOs:l 115, 17, 19, and 20 (e.g., a target-hybridizing sequence comprising or consisting of a sequence selected from SEQ ID NOs:ll-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof). In some embodiments, a capture probe oligomer comprises or consists of a sequence selected from SEQ ID NOs:l-5, 7, 9, and 10. In some embodiments, the oligomer combination includes at least two capture probe oligomers (e.g., at least two capture probe oligomers as above). A first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 19 (e.g., a capture probe oligomer comprising the sequence of SEQ ID NO:9) and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO;20 (e.g., a capture probe oligoiner comprising the sequence of SEQ ID NO: 10) are particularly suitable for use together in oligomer combinations as described herein.

In certain variations, an oligomer combination as described herein further comprises at least one détection probe oligomer confîgured to specifïcally hybridize to a Plasmodium sp. target sequence that is amplifîable using the at least two amplification oligomers targeting a Plasmodium sp. target région. In some embodiments where a Plasmodium sp. target région corresponds to a région of SEQ ID NO:180 from about nucléotide position 844 or about nucléotide position 910 to about nucléotide position 1038, about nucléotide position 1051, about nucleotîde position 1060, or about nucléotide position 1077, the oligomer combination includes a détection probe oligomer that specifïcally hybridizes to a target région corresponding to a région of SEQ ID NO:18ü from about nucléotide position 951 to about nucléotide position 998 or the full complément thereof. For example, a détection probe oligomer may include a targethybridizing sequence that is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 196 or ifs complément and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments thereof. In more spécifie variations, a détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément and (ii) includes the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments thereof. In other variations, a détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 196 or its complément and (ii) includes a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178, including compléments thereof. Particularly suitable détection probe oligomer target-hybridizing sequences include SEQ ID NOs:131, 132, 135, 140, 145, 147157, and 159-161, including compléments thereof. Suitable détection probes further include DNA équivalents and DNA/RNA chimerics of any of the above.

In some embodîments where a Plasmodium sp. target région corresponds to a région of SEQ ID NO: 180 from about nucléotide position 1153, about nucléotide position 1169, or about nucléotide position 1182 to about nucléotide position 1327, about nucléotide position 1354, or about nucléotide position 1382, the oligomer combination includes a détection probe 5 oligomer that specifically hybridizes to a target région corresponding to a région of SEQ ID NO:180 from about nucléotide position 1210 to about nucléotide position 1233 or the fiill complément thereof. For example, a détection probe oligomer may include a target-hybridizing sequence that is at least about 13 nucléotides in length and is (i) contained in the sequence of SEQ ID NO:I89 or its complément and (ii) includes a sequence selected from SEQ ID NO:190 and SEQ ID NO: 191, including compléments thereof. Particularly suitable détection probe oligomer target-hybridizing sequences include SEQ ID NOs: 125-130 and 143, including compléments thereof. Suitable détection probes further include DNA équivalents and DNA/RNA chimerics of any of the above.

Table 3 shows exemplary combinations of détection probe target hybridizing 15 sequences together with first and second amplification oligomer target-hybridizing sequences (Amp 1 and Amp 2) for détection of Plasmodium species target nucleic acid.

Table 3. Exemplary Combinations of Amplification Oligomer and Détection Probe Targethybridizing Sequences.

Amplification Oligomer THSs	Détection Probe THS (SEQ ID NO)
Amp 1 (SEQ ID NO)	Amp 2 (SEQ ID NO)
30	82	151
30	82	157
33	82	155
49	82	150
49	82	155
21	89	148
21	89	152
30	89	148
30	89	152
33	89	158
49	89	150
21	92	148
21	92	152
30	92	148
30	92	152
21	94	148
21	94	152
34	94	148
34	94	152

34	94	157
53	94	148
53	94	152
53	94	157
21	95	148
21	95	152
34	95	148
34	95	152
34	95	157
53	95	148
53	95	152
53	95	157
183	182	126
183	182	127
183	182	128
183	182	143
183	182	129
184	182	126

In some variations, an oligomer combination includes at least two détection probe oligomers (e.g., at least two spécifie détection probes as described herein). For example, where a Plasmodium sp. target région corresponds to a région of SEQ ID NO: 180 from about nucléotide position 844 or about nucléotide position 910 to about nucléotide position 1038, about nucléotide position 1051, about nucléotide position 1060, or about nucléotide position 1077, an oligomer combination may include (A) a First détection probe oligomer comprising a target-hybridizing sequenee that is (i) contained in the sequenee of SEQ ID NO: 197 or its complément and (ii) includes the sequenee of SEQ ID NO; 175 or its complément, and (B) a second détection probe oligomer comprising a target-hybridizing sequenee that is (î) contained in the sequenee of SEQ ID NO: 197 or its complément and (ii) includes the sequenee of SEQ ID NO:176 or its complément (including DNA équivalents or DNA/RNA chimerics of the foregoing). Particularly suitable combinations of first and second détection probe oligomers include a first détection probe oligomer comprising the target-hybridizing sequenee of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and a second détection probe oligomer comprising a target-hybridizing sequenee selected from SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

A détection probe oligomer may contain a 2'-methoxy backbone at one or more linkages in the nucleic acid backbone. In some embodiments, the at least one détection probe oligomer is provided in an amplicon détection reaction mixture.

Typically, a détection probe oligomer in accordance with the présent invention further încludes a label. Particularly suitable labels include compounds that émit a détectable light signal, e.g., fluorophores or luminescent (e.g·, chemilumînescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label, may be présent on a partîcular probe, or détection may rely on using a mixture of probes in which each probe is labeled with a compound that produces a détectable signal (see, e.g., US Pat. Nos. 6,180,340 and 6,350,579, each incorporated by reference herein). Labels may be attached to a probe by various means including covalent linkages, chélation, and ionic interactions, but preferably the label is covalently attached. For example, in some embodiments, a détection probe has an attached chemiluminescent label such as, e.g., an acridinium ester (AE) compound (see, e.g., US Patent Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744; each incorporated by reference herein), which in typical variations îs attached to the probe by a non-nucleotide linker (see, e.g., US Patent Nos. 5,585,481; 5,656,744; and 5,639,604, particularly at column 10, line 6 to column 11, line 3, and Example 8; each incorporated by reference herein). In other embodiments, a détection probe comprises both a fluorescent label and a quencher, a combination that is particularly usefiil in fluorescence résonance energy transfer (FRET) assays. Spécifie variations of such détection probes include, e.g., a TaqMan détection probe (Roche Molecular Diagnostics) and a molecular beacon (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; US Patent Nos. 5,118,801 and 5,312,728; each incorporated by reference herein).

A détection probe oligomer in accordance with the présent invention may further include a non-target-hybridizing sequence. Spécifie embodiments of such détection probes include, for example, probes that form conformations held by intramolecular hybridization, such as conformations generally referred to as hairpins. Particularly suitable hairpin probes include a molecular torch (see, e.g., US Patent Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945, each incorporated by reference herein) and a molecular beacon (see, e.g., Tyagi et al., supra; US 5,118,801 and US 5,312,728, supra). Methods for using such hairpin probes are well-known in the art.

In yet other embodiments, a détection probe is a linear oligomer that does not substantially form conformations held by intramolecular bonds. In spécifie variations, a linear détection probe oligomer includes a chemiluminescent compound as the label, preferably an acridinium ester (AE) compound.

Also provided by the présent invention are détection probe oligomers, capture probe oligomers, and combinations thereof as described herein.

In other aspects, the présent invention provides methods for detecting the presence or absence of Plasmodium sp. in a sample from a subject. Such methods generally inciude performing a nucleic-acid-based détection assay for the spécifie détection in the sample of Plasmodium sp. nucleic acid. A nucleic-acid-based détection assay for spécifie détection of Plasmodium sp. may use any one or more Plasmodium sp.-specific oligomers as described herein (e.g., an oligomer combination as described herein comprisîng at least two amplification oligomers; or a détection probe or combination of détection probes as described herein). A positive signal from a nucleic-acid-based détection assay in accordance with the présent invention is indicative of the presence of one or more of Plasmodium falciparum, Plasmodium knowlesi. Plasmodium malariae, Plasmodium ovale and/or Plasmodium vivax in a sample.

In some embodiments of a method comprisîng the use of a nucleic-acid-based détection assay, an ampli fication-based assay is used to detect Plasmodium sp. Such ampli fication-based assay methods generally inciude performing a nucleic acid amplification of an Plasmodium sp. target région and detecting the amplified product (e.g., by specifîcally hybridizing the amplified product with a nucleic acid détection probe that provides a signal to indicate the presence of Plasmodium sp. in the sample). The amplification step includes contacting the sample with one or more amplification oligomers spécifie for a target sequence in a Plasmodium sp. target nucleic acid to produce an amplified product if Plasmodium sp. nucleic acid is présent in the sample. In particular embodiments, a combination of at least two amplification oligomers as described herein are used at the amplification step. Amplification synthesizes additional copies of the target sequence or its complément by using at least one nucleic acid polymerase and an amplification oligomer to produce the copies from a template strand (e.g., by extending the sequence from a primer using the template strand). Suitable amplification methods inciude, for ex ample, replicase-mediated amplification, polymerase chaîn reaction (PCR), ligase chaîn reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). Such amplification methods are well-known in the art (see, e.g., discussion of amplification methods in Définitions section, supra) and are readily used in accordance with the methods of the présent disclosure.

Détection of the amplified products may be accomplished by a variety of methods to detect a signal specifîcally associated with the amplified target sequence. The nucleic acids may be associated with a surface that results in a physical change, such as a détectable electrical change. Amplified nucleic acids may be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g, an intercalating agent such as ethidium bromide or cyber green), or detecting an increase in dye associated with nucleic acid in solution phase. Other methods of détection may use uses a hybridizing step that includes contacting the amplified product with at least one détection probe configured to specifically hybridize to a sequence in the amplified product and detecting the presence of the probe:product corn pl ex, or b y using a complex of probes that may amplify the détectable signal associated with the amplified products (e.g., US Patent Nos, 5,424,413; 5,451,503; and 5,849,481). Directly or îndirectly labeled probes that specifically associate with the amplified product provide a détectable signal that indicates the presence of the target nucleic acid in the sample. In some embodiments, a method utilizing an amplification-based assay for détection of Plasmodium species utilizes one or more détection probe oligomers as described herein for détection of an amplified product.

Détection probes that hybridize to the complementary amplified sequences may be DNA or RNA oligomers, or oligomers that contain a combination of DNA and RNA nucléotides, or oligomers synthesized with a modified backbone, e.g., an oligomer that includes one or more 2'-methoxy substituted ribonucleotides. Probes used for détection of the amplified sequences may be unlabeled and detected indîrectly (e.g., by binding of another binding partner to a moiety on the probe) or may be labeled with a variety of détectable labels. In some embodiments of a method for detecting Plasmodium species, such as in certain embodiments using transcriptioumediated amplification (TMA), the détection probe is a linear chemîluminescently labeled probe such as, e.g., a linear acridinium ester (AE) labeled probe. The détection step may also provide additional information on the amplified sequence, such as, e.g., ail or a portion of its nucleic acid base sequence. Détection may be perfonned after the amplification reaction is completed, or may be performed sîmultaneously with amplifying the target région, e.g., in real time. In one embodiment, the détection step allows homogeneous détection, e.g., détection of the hybridized probe without removal of unhybridîzed probe from the mixture (see, e.g., US Patent Nos. 5,639,604 and 5,283, 174).

In embodiments that detect the amplified product near or at the end of the amplification step, a linear détection probe may be used to provide a signal to indicate hybridization of the probe to the amplified product. One example of such détection uses a luminescentally labeled probe that hybridizes to target nucleic acid. Luminescent label is then hydrolyzed from non -hybridized probe. Détection is performed by chemilummescence using a luminometer (see, e.g., International Patent Application Pub. No. WO 89/002476). In other embodiments that use real-time détection, the détection probe may be a hairpin probe such as, for example, a molecular beacon, molecular torch, or hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to amplified product. Such probes may comprise target-hybridizing sequences and non-target-hybrîdizing sequences. Varions forms of such probes hâve been described prevîously (see, e.g., US Patent Nos. 5,118,801 ; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1).

Some amplification methods that use TM A amplification include the following steps. Briefly, the target nucleic acid that contains the sequence to be ampli fi ed is provided as single stranded nucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art will appreciate that conventional melting of double stranded nucleic acid (e.g., dsDNA) may be used to provide single-stranded target nucleic acids. A promoter primer binds specifically to the target nucleic acid at its target sequence and a reverse transcriptase (RT) extends the 3' end of the promoter primer using the target strand as a template to create a cDNA copy of the target sequence strand, resulting in an RNA:DNA duplex. An RNase digests the RNA strand of the RNA:DNA duplex and a second primer binds specifically to its target sequence, which is located on the cDNA strand downstream from the promoter primer end. RT synthesizes a new DNA strand by extending the 3' end of the second primer using the first cDNA template to create a dsDNA that contains a functional promoter sequence. An RNA polymerase spécifie for the promoter sequence then initiâtes transcription to produce RNA transcripts that are about 100 to 1000 amplified copies (amplicons) of the initial target strand in the reaction. Amplification continues when the second primer binds specifically to its target sequence in each of the amplicons and RT créâtes a DNA copy from the amplicon RNA template to produce an RNA:DNA duplex. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA duplex and the promoter primer binds specifically to its complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer to create a dsDNA that contains a functional promoter to which the RNA polymerase binds to transcribe addîtional amplicons that are complementary to the target strand. The autocatalytic cycles of making more amplicon copies repeat during the course of the reaction resulting in about a billion-fold amplification of the target nucleic acid présent in the sample. The amplified products may be detected in real-time during amplification, or at the end of the amplification reaction by using a probe that binds specifically to a target sequence contained in the amplified products. Détection of a signal resulting from the bound probes indicates the presence of the target nucleic acid in the sample.

In some embodiments, the method utîlizes a reverse TMA reaction. In such variations, the initial or forward amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid in the vicînity of the 3'-end of the target région. A reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3'-end of the primer using the target nucleic acid as a tempiate. The second or reverse amplification oligomer is a promoter primer or promoter pro vider having a target-hybridizing sequence configured to hybridize to a target-sequence contained within the synthesized cDNA strand. Where the second amplification oligomer is a promoter primer, RT extends the 3' end of the promoter primer using the cDNA strand as a tempiate to create a second. cDNA copy of the target sequence strand, thereby creating a dsDNA that contains a functional promoter sequence. Amplification then continues essentially as described above for initiation of transcription from the promoter sequence utilizing an RNA polymerase. Altematively, where the second amplification oligomer is a promoter provider, a terminating oligonucleotide, which hybridizes to a target sequence that is in the vicinity to the 5'end of the target région, is typically utilized to terminale extension of the priming oligomer at the 3'-end of the tenninating oligonucleotide, thereby providing a defmed 3'-end for the initial cDNA strand synthesized by extension from the priming oligomer. The target-hybridizing sequence of the promoter provider then hybridizes to the defined 3'-end of the initial cDNA strand, and the 3'-end of the cDNA strand is extended to add sequence complementary to the promoter sequence of the promoter provider, resulting in the formation of a double-stranded promoter sequence. The initial cDNA strand is then used a tempiate to transcribe multiple RNA transcripts complementary to the initial cDNA strand, not including the promoter portion, using an RNA polymerase that recognizes the double-stranded promoter and initiâtes transcription therefrom. Each of these RNA transcripts is then available to serve as a tempiate for further amplification from ihe first priming amplification oligomer.

In some embodiments of a method comprising the use of a nucleic-acid-based détection assay, a non-amplification-based assay is used to detect Plasmodium sp. In some such embodiments, the non-amplification-based assay is a hybridization assay comprising the hybridization of a spécifie détection probe to a target nucleic acid. Methods for conducting polynucleotide hybridization assays hâve been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general bindîng methods known, including those referred to in, e.g., Maniatis et al, Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor, N.Y., 2002), and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academie Press, Inc., San Diego, Calif., 1987). Generally, the probe and sample are mixed under conditions that will permit spécifie nucleic acid hybridization, and spécifie hybridization of the probe to its respective target is then detected. Nucleic acid hybridization is adaptable to a variety of assay formats. One suitable format is the sandwich assay format, which is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support, which has adsorbed to it or covalent!y coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence. Target nucleic acid is hybridized to the immobilized probe, and a second, labeled détection probe - which is complementary to a second and different région of the same DNA strand to which the immobilized, unlabeled nucleic acid probe is hybridized - is hybridized to the [target nucleic acid]: [immobilized probe] duplex to detect the target nucleic acid. Another exemplary format utilizes electroChemical détection of target nucleic acids hybridized to unlabeled détection probes immobilized on a suitable electrode surface as a signal transducer. See, e.g., Drummond et al., Nat. Biotechnol. 21:1192, 2003; Goodîng, Electroanalysls 14: 1149, 2002; Wang, Anal. Chim. Acta 469:63, 2002; Cagnîn et al., Sensors 9:3122, 2009; Katz and Willner, Electroanalysls 15:913, 2003; Daniels and Pounnand, Electroanalysls 19: 1239, 2007.

In certain embodiments of a method for detecting Plasmodium species comprising a hybridization assay, the hybridization assay utilizes one or more détection probe oligomers as described herein.

In some embodiments, a non-amplification-based assay for détection of Plasmodium sp. is a cleavage-based assay, in which a probe oligonucleotide containing a non-target-hybridizing flap région is cleaved in an overlap-dependent manner by a flap endonuclease to release a cleavage product that is then detected. Exemplary cleavage-based assay reagents are described in, e.g., Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn. 4: 135144, 1999), and Allawi étal. (J. Clin. Microbiol. 44:3443-3447, 2006).

Appropriate conditions for flap endonuclease reactions are either known or can be readily determined using methods known in the art (see, e.g., Kaiser et al., J. Biol. Chem. 274:2138721394, 1999). Exemplary flap endonucleases that may be used in the method include Thermus aquaticus DNA polymerase I, Thermus thermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcus Jannaschii FEN-1, Pyrococcus fiiriosus FEN-1, Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1, CLEAVASE® (Hologic, Inc., Madison, WI), 5, cerevisiae RTH1, S. cerevisiae RAD27, Schizosaccharomyces pombe rad2, bactériophage T5 5' -3' exonuclease, Pyrococcus horikoshii FEN-1, human endonuclease 1, calf thymus 5-3' exonuclease, including homologs thereof in eubacteria, eukaryotes, and archaea, such as members of the class II famity of structure-specific enzymes, as well as enzymatically active mutants or variants thereof. Descriptions of flap endonucleases can be found in, for example, Lyamichev et al., Science 260:778-783, 1993; Eis et al., Nat. Biotechnol. 19:673-676, 2001; Shen et al., Trends in Bio. Sci. 23: 171 -173, 1998; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999; Ma et al., J. Biol. Chem. 275:24693-24700, 2000; Allawi et al., J. Mol. Biol. 328:537-554, 2003; Shanna et al., J. Biol. Chem. 278:23487-23496, 2003; and Feng et al., Nat. Struct. Mol. Biol. ! 1 :450-456, 2004.

In certain variations, a cleavage-based assay detects an RNA target nucleic acid of Plasmodium sp., and the cleavage-based assay utilizes a flap endonuclease that is capable of cleaving and RNA:DNA linear duplex structure. In some alternative embodiments, a cleavagebased assay detects a DNA target nucleic acid of Plasmodium sp., and the cleavage-based assay utilizes a flap endonuclease that is capable of cleaving and DNA:DNA linear duplex structure. Exemplary flap endonucleases capable of cleaving RNA:DNA duplexes include polymerasedeficient 5' nucleases of the genus Thermus as well as certain CLEAVASE® enzymes (Hologic, Inc., Madison, WI) such as, for example, CLEAVASE® BN (BstX-Notl délétion of Taq polymerase, see US Patent No. 5,614,402), CLEAVASE® II (AG mutant of full length Taq polymerase, see US Patent No. 5,614, 402), CLEAVASE® VII (synthesis-deficient mutation of full length Thermus thermophilus polymerase), CLEAVASE® IX (polymerase déficient mutant of the Tth DNA polymerase), and CLEAVASE® XII (polymerase déficient chimeric polymerase constructed from fragments of taq DNA polymerase and Tth DNA polymerase). Exemplary flap endonucleases capable of cleaving DNA:DNA duplexes include the flap endonucleases indicated above, as well as CLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1 (Archaeoglobus fulgidus FEN-1 with 6 histidines on the C-terminus), CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1 (Archaeoglobus veneficus FEN-1 with 6 histidines on the C-terminus).

In some embodiments, a cleavage-based assay detects an RNA target nucleic acid of Plasmodium sp., and the assay includes a step fbr synthesizing a DNA complément of an RNA target région, which cDNA strand is then hybridized to overlapping first and second probe oligonucleotides to form a linear duplex cleavage structure for cleavage by the flap endonuclease. Reaction conditions for synthesizing cDNA from an RNA template, using an RNA-dependent DNA polymerase (reverse transcriptase), are well-known in the art.

In certain embodiments utilizing a nucleic-acid-based détection assay, the method further includes purifying the Plasmodium sp. target nucleic acid from other components in the sample. Such purification may include methods of separatîng and/or concentrating organisms contained in a sample from other sample components. In particular embodiments, purifying the target nucleic acid includes capturîng the target nucleic acid to specifically or non-specifically separate the target nucleic acid from other sample components. Non-specific target capture methods may involve sélective précipitation of nucleic acids from a substantially aqueous mixture, adhérence of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture that contains Plasmodium sp. nucleic acid and other sample components. In some embodiments, purification includes lysîng a sample of cells such as, for example, blood cells (e.g., red blood cells) and purifyîng any Plasmodium sp. target nucleic acid from the lysed cell sample. Exemplary lysis reagents and methods for used in accordance with the présent invention are described in US Pat. No. 10,093,989 and PCT Pub. No. WO 2017/1 89746, each incorporâted by reference herein.

In some embodiments, a target nucleic acid of Plasmodium sp. is separated from other sample components b y hybridizing the target nucleic acid to a capture probe oligomer. The capture probe oligomer comprises a target-hybridizing sequence configured to specifically or non-specifically hybridize to a target nucleic acid so as to form a [target nucleic acid]: [capture probe] complex that is separated from other sample components. Capture probes comprising target-hybridizing sequences suitable for non-specific capture of target nucleic acids are described in, e.g., PCT Pub. No. WO 2008/016988. In some spécifie variations comprising target-hybridizing sequence(s) configured to specifically hybridize to a Plasmodium sp. target nucleic acid, a Plasmodium sp.-spécifie capture probe comprises a target-hybridizing sequence that is up to about 30 contiguous nucléotides in length and includes a sequence substantially corresponding to a sequence selected from SEQ ID NOs: 11-15, 17, 19, and 20 (e.g., a targethybridizing sequence comprising or consisting of a sequence selected from SEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof). In a preferred variation, the capture probe binds the [target nucleic acid]:[capture probe] complex to an immobîlized probe to form a [target nucleic acid]:[capture probe]: [iminobilized probe] complex that is separated from the sample and, optionaily, washed to remove non-target sample components (see, e.g., US Patent Nos. 6,110,678; 6,280,952; and 6,534,273). In such variations, the capture probe oligomer further comprises a sequence or moiety that binds the capture probe, with its bound target sequence, to an immobîlized probe attached to a solid support, thereby permitting the hybridized target nucleic acid to be separated from other sample components.

In more spécifie embodiments, the capture probe oligomer includes a tail portion (e.g., a 3' tail) that is not complementary to target nucleic acid but that specifically hybridizes to a sequence on the immobîlized probe, thereby serving as the moiety allowing the target nucleic acid to be separated from other sample components, such as previously described in, e.g., U.S. Patent No. 6,110,678. Any sequence may be used in a tail région, which is generally about 5 to 50 nt long, and preferred embodiments include a substantially homopolymeric tail of about 10 to 40 nt (e.g., A10 to A40), more preferably about 14 to 33 nt (e.g., A14 to A30 or T3A14 to T3A30), that bind to a complementary immobîlized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle. In some such embodiments comprising target-hybridizing sequence(s) configured to specifically hybridize to Plasmodium sp. target nucleic acid, a Plasmodium sp.-spécifie capture probe comprises or consists of a nucléotide sequence selected from SEQ ID NOs:l-5, 7, 9, and 10.

Target capture typically occurs in a solution phase mixture that contains one or more capture probe oligomers that hybridize to the target nucleic acid under hybridizing conditions, usually at a température higher than the Tm of the [tail sequence]:[immobilized probe sequence] duplex. For embodiments comprising a capture probe tail, the [target nucleic acid]:[capture probe] complex is captured by adjusîing the hybridization conditions so that the capture probe tail hybridizes to the immobilized probe, and the entire complex on the solid support is then separated from other sample components. The support with the attached [immobilized probe] : [capture probe]: [target nucleic acid] may be washed one or more times to further remove other sample components. Preferred embodiments use a particulate solid support, such as paramagnetic beads, so that particles with the attached [target nucleic acid]:[capture probe]:[immobilized probe] complex may be suspended in a washing solution and retrieved from the washing solution, preferably by using magnetic attraction. In embodiments of the method comprising the use of an amplification-based détection assay, to limit the number of handling steps, a target nucleic acid may be amplified by simply mixing the target nucleic acid in the complex on the support with amplification oligomers and proceeding with amplification steps.

In accordance with the présent dîsclosure, detecting the presence or absence of Plasmodium sp. may be perfonned separately (e.g., in a separate reaction vessel), or performed together with another assay as a multiplex reaction System. Accordingly, in some embodiments, a method as described herein utilizes a multiplex reaction, where the reaction mix contains reagents for assaying multiple (e.g., at least two, three, four, or more) different target sequences in parallel. In these cases, a reaction mix may contain multiple different target-specific oligonucleotides for performing the détection assay. For example, in a method utilizing an amplification-based détection assay, a multiplex reaction may contain multiple sets (e.g., multiple pairs) of amplification oligomers (for example, multiple pairs of PCR primers or multiple pairs of TMA amplification oligomers (e.g., for TM A, multiple pairs of promot er primer and non-promoter primer, or multiple pairs of promoter provider and non-promoter primer)). In other embodiments utilizing a cleavage-based détection assay, a multiplex reaction may contain multiple probe oligonucleotides having different flaps, multiple different overlapping probe oligonucleotides, and multiple different FRET cassettes for detecting the different flaps, once they are cleaved.

The oligomer combination described herein may be in the form of a reaction mixture or a kit comprising the oligomers. The reaction mixture or kit may further include a number of option al components such as, for example, capture probe nucleic acids or arrays of capture probe nucleic acids. For an amplification reaction mixture, the reaction mixture will typically include other reagents suitable for perfonning in vitro amplification such as, e.g., buffers, sait solutions, appropriaie nucléotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and U TP), and/or enzymes (e.g., reverse transcriptase, and/or RNA polymerase), and will typically include test sample components, in which a Plasmodium sp. target nucleic acid may or may not be présent. A kit comprising an oligomer combination for amplification of Plasmodium sp. may also include other reagents suitable for performing in vitro amplification such as, e.g., buffers, sait solutions, appropriaie nucléotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNA polymerase). For an oligomer combination (e.g., reaction mixture or kit) that includes a détection probe together with an amplification oligomer combination targeting a common target nucleic acid, sélection of amplification oligomers and détection probe oligomers are linked by a common target région (i.e., the combination will include a probe that binds to a sequence amplifîable by the amplification oligomer combination).

The compositions, methods, reaction mixtures, Systems, kits and the like for détection of Plasmodium nucleic acids are further illustrated by the following non-limiting examples.

EXAMPLES

Parasite Transport Solution generally refers to a solution formulated to preserve a sample, and in some instances formulated to at least partially lyse one or more cell types in a sample. One exemplary parasite transport solution comprises 15 mM sodium phosphate monobasic, 15 mM sodium phosphate dibasîc, 1 mM EDTA, 1 mM EGTA, and 110 mM lithium lauryl sulfate (LLS), at pH 6.7. Another exemplary parasite transport solution comprises an aqueous solution of 100 mM TRIS, 30 mM magnésium chloride, and 6% (v/v) LLS, at pH 7.5. A further exemplary parasite transport solution comprises an aqueous solution of 14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v/v) LLS, and 0.1 mM EDTA, at a pH of 7.4. Other formulations of parasite transport solutions may function equally well.

Target Capture Reagent generally refers to a solution containing a number of components that facîlitate capture of a nucleic acid from a solution. One exemplary Target Capture Reagent comprises 250 mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mM EDTA, at pH 6.4, and 250 pg/mî of magnetic particles (1 micron SERA51

MAG^ATM MG-CM particles, GE Healthcare Lifesciences) with dT₁₄ oligomers covalently bound thereto. Another exemplary Target Capture Reagent comprises 790 mM HEPES, 453 mM lithium hydroxide, 10% w/v LLS, 230 mM Succinic Acid, 0.03% w/v Foam Ban MS-575, and 0.0125% w/v of magnetic particles (1 micron SERA-MAG^ATM MG-CM particles, GE Healthcare Lifesciences) with dT^ oligomers covalently bound. Other formulations of Target Capture Reagent may fonction equally as well.

Wash Solution generally refers to a solution containing 10 mM HEPES, 150 mM sodium chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v/v) éthanol, 0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, and 0.1% (w/v) sodium lauryl sulfate, at pH 7.5.

Probe Reagent generally refers to a solution containing one or more labeled détection probes. One exemplary Probe Reagent îs a solution made up of from about 75 to about 100 mM lithium succinate, 2% (w/v) LLS, 15 mM mercaptoethanesulfonate, 1.2 M lithium chloride, 20 mM EDTA, and 3% (v/v) éthanol, at pH 4.7. Another exemplary Probe Reagent is a solution made up of from about 75 to about 100 mM succinic acid, 3.5% (w/v) LLS, 75 mM lithium hydroxide, 15 mM aldrithiol-2, LO M lithium chloride, 1 mM EDTA, and 3.0% (v/v) éthanol, at pH 4.1-4.3. Other formulations may perforai equally as well.

Amplification Reagent generally refers to a concentrated mixture of reaction components to facilitate amplification reactions. An Amplification Reagent will comprise a number of different reagents at varions concentrations dependîng on factors such as for example amplification type (PCR, TM A, etc.), target nucleic acids (GC content), and the like. One exemplary Amplification Reagent comprises 47.6 mM Na-HEPES, 12.5 mM N-acetyl-Lcysteine, 2.5% TRITON™ X-100, 54.8 mM KC1, 23 mM MgC12, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 7.06 mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM Na2EDTA, 5% v/v glycerol, 2.9% trehalose, 0.225% éthanol, 0.075% methylparaben, 0.015% propylparaben, and 0.002% Phénol Red, at pH 7.5-7.6. Another exemplary Amplification Reagent comprises 19.1 mM Trizma Base, 7.5mM Trizma Hydrochloride, 23.3 mM KC1, 21.5 mM MgC12, 1 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 6.5 mM rATP, 4.0 mM rCTP, 4.0 mM UTP, 6.5 mM rGTP, 3.33% v/v glycerol, 0.05 mM Zinc Acetate, 6 ppm Pro Clin 300 preservative, at pH 8.25-8.45. Other formulations of amplification reagent may fiinctîon equally well. Primers may be added to the amplification reagent or added to amplification reactions separate from the amplification reagent. Enzymes in an amplification reagent can include one or more of Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) and bactériophage T7 RNA polymerase for which units are functionally defmed as: 1 U of MMLV-RT incorporâtes 1 nmol of dTTP in 10 min at 37C using

200-400 micromolar oligo dT-primed poly(A) as template, and 1 U of T7 RNA polymerase incorporâtes 1 nmol of ATP into RNA in 1 hr at 37C using a DNA template containing a T7 promoter.

Hybridization Reagent generally refers to a solution made up of reagents having concentrations in the range of about: 75-100 mM succinic acid, 2%-3.5% (w/v) LLS, 75-100 mM lithium hydroxide, 14-16 mM aldrithioI-2, 1.0-1.2 M lithium chloride, 20-1000 mM EDTA, and 2.0-4.0% (v/v) éthanol, at pH 4-5 Other formulations for a Hybridization Reagent may fonction equally well.

Sélection Reagent generally refers to a solution containing 600 mM boric acid, 182.5 mM sodium hydroxide, 1% (v/v) octoxynol (TRITON® X-I00), at pH 8.5.

Détection Reagents include Detect Reagent I, which generally refers to a solution containing 1 mM nitric acid and 32 mM hydrogen peroxide, and Detect Reagent Π, which generally refers to a solution of 1.5 M sodium hydroxide.

Example 1

Primer screening was performed using Transcription-Médiated Amplification (TMA) on the manual Procleix Enhanced Semi-automated System (eSAS) using Plasmodium falciparum in vitro transcript (IVT) . An assay rack consisted of 10 rows of Ten-tube units (TTUs). Seventy five microliters (75 pL) of Amplification Reagent and 5 picomoles of each T7 promoter provider oligonucleotide and non-T7 primer oligonucleotide were added to the appropriate tubes on the rack such that each combination of amplification oligomers were tested with three replicates of P. falciparum IVT at 30 and 10 copies per reaction and two replicates of B. microti IVT at 1,000,000 copies per réaction, where applicable. B. microti was included in initial screening as a cross reactivity specimen due to the conserved régions between Babesia and Plasmodium. It is necessary to détermine that amplification and détection Systems are spécifie to Plasmodium. To achieve the target copies per reaction, 10 pL of P. falciparum IVT at 3 c/pL or 1 c/pL diluted in a buffer was spiked into the appropriate tubes, and 10 pL of B. microti IVT at 100,000 c/pL diluted in a buffer were spiked into the appropriate tubes. Various combinations of primers were tested. This set-up allows for 10 primer combinations to be tested per rack. Once the primer combinations and IVTs were spiked, 200 pL of oil was added to each tube and then the rack was covered with sealing cards and vortexed for a minimum of 20 seconds.

The rack was then incubated in a water bath at 60±l°C for 10±l minutes followed by incubation in a 41.5±1°C water bath between 9 and 20 minutes. While the rack remained in the water bath, the sealing cards were removed and 25 pL of commercially available Procleix Ultrio 53

Plus enzyme reagent (Grifols Diagnostic Solutions Inc.) was added to each reaction tube and then covered agam with sealing cards. The rack was gently shaken to mix and then covered again with sealing cards and incubated for another 60±5 minutes in the 4] ,5±l °C water bath.

After incubation completed, the rack was transferred to the hybridization protection assay (HPA) area where the sealing cards were removed. 1 OOpL of Probe reagent consisting of an Acridinium-Ester (AE) labeled probe added at a total desired concentration of at least 2.5e6 Relative Light Units (RLU) per reaction to a Hybridization reagent. Probe reagent was then added to the appropriate reaction tubes. The tubes were covered with sealing cards and the rack was vortexed for a minimum of 20 seconds after which the rack was incubated in a water bath at 61±2°C for 15±1 minutes.

The rack was removed from the water bath, the sealing cards removed, and 250 pL of commercîally avaîlable Procleix Ultrio Plus Sélection reagent (Grifols Diagnostic Solutions Inc.) was added to each tube. The tubes were covered with sealing cards and vortexed for a minimum of 20 seconds and then retumed to the 61±2°C water bath and incubated for 10±l minutes. After incubation, the rack was allowed to cool in a 23±4°C water bath for a minimum of 10 minutes.

For détection the TTUs are removed from the rack and loaded on to the automated Leader instrument for subséquent light off using commercîally avaîlable Procleix Auto Detect 1 and 2 reagents (Grifols Diagnostic Solutions Inc.) and the results were exported for analysis of the signal in Relative Light Units (RLU).

AE-labeled probes screened in this example are shown in Table 4 below. Probes were scrcened wîth amplification oligomer pairs in four experimental groups: Group 1, Groups 2a and 2b, and Group 3.

Table 4.

Probe #	SEQ ID NO:	2MeAE linker site
1	159	7,8
3	159	9,10
4	151	6,7
5	151	7,8
6	151	8,9
7	150	8,9
8	155	7,8
12	160	13,14
14	147	11,12
15	157	10,11
16	156	10,11
20	158	6,7

Probe #	SEQ ID NO:	2MeAE linker site
21	158	7,8
22	158	10,11

Group L Probes 1, 3, 4-6, 12, and 14-16 were each tested with SEQ ID NO:59 (T7 promoter pro vider oligomer) and SEQ ID NO:30 (non-T7 oligomer). The results of this probe screen are shown in Table 5 below. Candidates showed no cross-reactivity with B. microti IVT at 5 le6 copies per reaction. In addition, probes having the same nucléotide sequence but with different 2MeAE linker sites (see, e.g., probes 1 and 3; see also probes 4, 5, and 6) performed well irrespective of their different labelling.

Table 5.

	P. fal 30 c/rxn	P. fal 30 c/rxn	P. fal 30 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	B.mie le6 c/rxn	B.mic le6 c/rxn	B.mic le6c/c/rxn
Probe 1	1,222,972	1,157,299	1,201,553	927,342	1,093,944	98,711	919	985	871
Probe 3	1,179,477	1,192,980	1,155,140	1,130,568	854,803	1,206,816	1,515	1,640	1,708
Probe 5	816,162	815,694	774,947	708,438	745,288	519,080	1,562	1,253	1,322
Probe 4	965,152	1,019,364	1,043,712	767,525	957,437	905,300	1,203	1,565	1,276
Probe 6	1,179,590	1,182,350	1,136,391	1,034,114	1,098,623	856,584	2,211	3,484	2,473
Probe 12	1,801,146	1,739,366	1,606,426	698,553	1,610,398	1,515,342	1,540	1,278	2,523
Probe 14	1,017,636	997,356	1,015,960	951,505	830,243	909,076	870	865	1,470
Probe 15	1,116,536	1,203,192	1,038,531	1,093,438	958,900	891,744	1,174	1,019	1,803
Probe 16	182,425	195,626	73,184	141,723	47,725	52,841	4,155	2,440	2,629
NEG	Probe 1	Probe 3	Probe 4
1,108	1,046	1,310	1,935	1,749	1,784	1,319	1,489	1,388
Probe 4	Probe 6	Probe 12
2,614	1,194	2,321	2,673	2,935	4,364	1,473	1,474	2,528
Probe 14	Probe 15	Probe 16
1,311	1,597	978	1,359	1,171	1,475	3,940	1,430	1,869

Groups 2a and 2b. In Group 2a, the following primer/probe combinations were tested: each of probes 8 and 20-22 paired with SEQ ID NO:59 and SEQ ID NO:33 (T7/NT7), each of probes 7 and 20-22 paired with SEQ ID NO:59 and SEQ ID NO:52 (T7/NT7), and each of probes 7 and 8 paired with SEQ ID NO:59 and SEQ ID NO:49 (T7/NT7). In Group 2b, the 15 following primer/probe combinations were tested: each of probes 20-22 paired with SEQ ID

NO:66 and SEQ ID NO:33 (T7/NT7), each of probes 7 and 20-22 paired with SEQ ID NO:66 and SEQ ID NO:52 (T7/NT7), and probe 7 paired with SEQ ID NO:66 and SEQ ID NO:49 (T7/NT7).

The results of this probe screen are shown in Tables 6 and 7 below. Systems with the non-T7 oligomer of SEQ ID NO:52 did not amplify Plasmodium. While Systems with the T7/NT7 oligomer pair of SEQ ID NO:59/SEQ ID NO:33 paired with any of Probes 20-22 dîd not detect Plasmodium, these probes detect Plasmodium when used with the T7/NT7 oligomer 5 pair of SEQ ID NO:66/SEQ ID NO:33.

Table 6.

T7/NT7 + Probe	P. fai 30 c/rxn	P. fal 30 c/rxn	P. fal 30 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	B. mtc le6 c/rxn	B. mic le6 c/rxn	NEG	NEG
59/49 +Probe 7	1,414,377	1,429,342	1,423,366	1,293,402	1,271,942	1,347,111	1,967	1,113	1,651	1,603
59/52 +P robe 7	15,998	92,089	4,310	676	1,465	3,151	1,473	3,105	793	1.315
59/49 +Probe 8	1,556,915	1,585,955	1,594,418	1,523,106	1,419,616	1,564,211	7,048	2,935	3,710	5,601
59/33 +Probe 8	1,026,164	1,188,939	1,511,173	1,022,388	1,030,495	421,293	2,804	2,963	4,303	6,879
59/33 +Probe 20	438,708	543,560	635,481	385,704	410,210	728,374	1,263	1,475	1,151	1,295
59/52 +Probe 20	1,058	1,196	875	9,308	69,190	686	926	930	1,169	1,771
59/33 +Probe 21	1,131,775	1,206,597	957,441	1,558	260,318	653,321	1,217	3,177	1,160	2,281
59/52 +Probe 21	2,260	8,505	1,529	901	1,109	2,917	1,314	1,386	1,91 i	1,978
59/33 +P robe 22	475,040	486,044	474,661	342,995	713	427,613	1,257	729	585	1,143
59/52 +Probe 22	53,691	57,052	1,205	1,225	2,526	2,948	4,203	677	5,728	1,344

Table 7.

T7/NT7 + Probe	P. fal 30 c/rxn	P. fal 30 c/rxn	P. fal 30 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	B. mic le6 c/rxn	B. mic le6 c/rxn	NEG	NEG
66/49 + Probe 7	1,697,508	1,654,623	1,702,775	1,632,642	1,670,622	1,710,421	4,352	6,899	1,718	780
66/52 + Probe 7	698,772	9,692	1,394,770	1,732	926	198,284	3,588	4,249	1,173	890
66/33 + Probe 20	2,085,094	1,917,183	1,977,048	2,166,352	1,898,001	1,529,882	1,294	1,645	2,378	1,368
66/52 + Probe 20	5,387	6,054	1,395,984	78,381	13,600	1,825	2,979	5,688	5,636	2,165
66/33 + Probe 21	2,787,188	2,641,943	2,691,394	2,189,645	1,834,205	2,528,97]	5,627	3,547	3,817	4,417
66/52 + Probe 21	103,016	1,944,005	8,745	2,694,902	1,167,241	5,856	3,229	4,437	6,515	2,704

66/33 + Probe 22	1,976,930	2,048,269	2,206,921	2.248.244	1,576,131	1,897,000	1,619	5,010	2,252	2,978
66/52 + Probe 22	314,430	1,222,503	36,814	1,596	60,021	1 10,944	1,831	2,930	2,213	1,337

Group 3. Re-designs of the non-T7 primer of SEQ ID NO:33 were tested. Each of probes 7 and 8 were paired with each of SEQ ID NO:66/SEQ ID NO:25 and SEQ ID NO:66/SEQ ID NO:35 (T7/NT7). In addition, probe 20 was paired with SEQ ID NO:66/SEQ ID NO:33 5 (T7/NT7).

The results of this probe screen are shown in Table 8 below. The T7/NT7 oligomer pair of SEQ ID NO66/SEQ ID NO:33 perfonned well with probe 20. Redesigns for the non-T7 oligomer of SEQ ID NO:33 (SEQ ID NO;25 and SEQ ID NO:35) induced false positives with probes 7 and 8.

Table 8.

T7/NT7 + Probe	P. fai 30 c/rxn	P. fai 30 c/rxn	P. fal 30 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	P. fal 10 c/rxn	NEG	NEG	NEG	NEG
66/33 +Probe 20	2,259,738	2,251,254	2,259,806	2,193,185	2,229,852	2,305,634	10,987	2,278	2,517	833
66/25 +Probe 7	1,639,345	1,634,590	1,094,963	1,577,109	1,447,969	1,443,799	659,001	907,064	955,394	106,595
69/25 +Probe 7	1,604,438	1,518,505	1,606,930	1,473,808	1,431,398	1,542,777	873,665	963,803	849,156	705,831
66/35 +Probe 7	1,590,743	1,608,076	1,615,693	1,595,727	1,351,299	1,582,092	759,553	622,876	495,724	743,057
69/35 +Probe 7	1,669,062	1,684,886	1,610,911	1,604,953	1,279,697	1,584,979	816,245	874,237	467,269	1,302,044
66/25 +Probe 8	1,690,077	1,673,850	1,597,686	1,601,896	1,359,389	1,572,372	1,172,325	693,937	286,919	536,015
69/25 +Probe 8	1,607,570	1,637,678	1,622,369	1,613,782	1,538,278	1,613,946	93,383	1,029,827	1,213,750	1,192,320
66/35 +Probe 8	1,622,258	1,609,624	1,572,273	1,600,610	1,570,232	1,611,927	1,235,255	947,640	841,628	1,143,229
69/35 +Probe 8	1,623,329	1,679,140	1,634,462	1,628,953	1,509,017	1,583,660	604,251	705,198	1,259,683	966,102

Example 2

This example describes materials and methods for screening of candidate amplification 15 Systems using TMA on the full automated Procleix Panther system (Grifols Diagnostic Solutions Inc.).

Specimens included P. falciparum, P. knowlesi, P. malariac, P. ovale, and/or P. vivax IVT diluted in buffer. Specimens may also include B. microti IVT as a cross-reactivity specimen. It is necessary to détermine that amplification and détection Systems are spécifie to Plasmodium. An assay calibrator comprising a P. falciparum IVT panel at 500 c/mL was included to détermine the analyte cutoff for the run. The assay software uses the analyte cutoff to détermine if samples are réactivé or ηοη-reactîve. Samples with a signal to cutoff ratio of > 1 are considered réactivé, while those < 1 are non-reactive. Assay Reagents used included the following: a Target Capture Reagent (TCR) comprising of at least one target capture oligomer (TCO); an Amplification reagent comprising at least one T7 promoter provider and at least one non-T7 primer; a Probe reagent consisting of at least one AE labeled probe; Ultrio Plus Enzyme reagent; and Sélection reagent.

Example 3: Plasmodium Target Capture Probe Screening

Candidate target capture probes (TCOs) were screened with samples containing P. falciparum IVT at 500 c/mL prepared in 3 mL Parasite Transport Medium (PTM; 100 mM TRIS, 30 mM magnésium chloride, and 6% (v/v) LLS, at pH 7.5) with 1 mL whole blood. TMA reactions were performed on the fully automated Procleix Panther System substantially as described in Example 2. TCOs of SEQ ID NOs. 1-5, 7, 9, and 10 were tested in reactions using T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol/rxn each), and détection probes of SEQ ID NO:148 and SEQ ID NO: 152 ( 1.9e6 RLU/rxn each).

Results are shown in Tables 9 and 10 below. Assay performance was best with TCOs SEQ ID NO:9 and SEQ ID NO: 10. SEQ ID NO:3 was not optimal in this assay.

Table 9.

TCO	Mean Total RLU*
SEQ ID NO:3	663,034
SEQ ID NO:1	873,914
SEQ ID NO:9	1,474,284
SEQ ID NO;2	985,039
SEQ ID NO: 10	1,455,387
SEQ ID NO;7	793,219
SEQ ID NO:5	951,504
SEQ ID NO:4	952,023

* N=10 for ail TCOs except SEQ ID NO:3 (N=20)

Table 10.

TCO	%CV Total RLU*
SEQ ID NO:3	19.2
SEQ IDNO:1	19.7
SEQ ID NO:9	2.6
SEQ ID NO:2	11.2
SEQ 1DNO:10	3.6
SEQ ID NO:7	28.1
SEQ ID NO:5	14.4
SEQ ID NO:4	14.4

* N=10 for ail TCOs except SEQ ID NO:3 (N=20)

Example 4: Analvtical Sensitivitv - LoD of RN A Copies/mL

Limît of détection (LoD) for a candidate amplification system was assessed by probit analysis using in vitro synthesized transcripts for P. falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax. TMA reactions were performed on the fully automated Procleix Panther system substantially as described in Example 2. The IVT for each species was serially diluted in buffer to 100, 30, 10, 3, 1 and 0 copies/mL and tested in 32 replicates for each level per species. Specimens were tested using TMA on the fully automated Procleix Panther system (Grifols Diagnostic Solutions Inc.). An assay calibrator comprisîng a P. falciparum IVT panel at 500 c/mL was included to détermine the analyte cutoff for the run. The assay software uses the analyte cutoff to détermine if samples are reactive or non-reactive. Samples with a signal to cutoff ratio of > 1 are considered réactivé, while those < 1 are non-reactive. For this assay, the foliowing oligomers were used: T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:2I and SEQ ID NO:30 (5 pmol/rxn each), détection probes of SEQ ID NO:148 and SEQ ID NO:152 (1.9e6 RLU/rxn each), and TCOs of SEQ ID NO:9 and SEQ ID NO: 10.

Results are shown in Table 11 below. Similar LoD values were observed for the five species tested. 95% LoD ranged from 9.4 to 14.8 copies/mL.

Table 11.

In vitro transcript (N=32)	50% LoD in Copies/mL (Fiducial Limits)	95% LoD Copies/mL (Fiducial Limits)
P. falciparum	2.1 (1.3-2.9)	9.4 (6.5-17.3)
P. knowlesi	3.3 (2.2-4.5)	14.8 (10.2-27.3)

P. malariae	3.3(2.2-4.4)	13.1 (9.1 -24.4)
P. ovale	2.5(1.5-3.5)	13.6 (9.0-27.8)
P, vivax	4.5 (3.4-5.7)	11.6(8.9-17.7)

Example 5: Analytical Sensitivitv - LoD of RNA Parasites/mL

Lîmit of détection (LoD) for a candidate amplification System was assessed by probit analysis using cultured parasite-infected cells. Specifically, conditions were tested using a lysed négative whole blood specimen and diluted cultured P. falciparumAnfecied érythrocytes. The cultured sample was receîved with a known percent parasitemîa and RBC count determined by Fluorescence-Activated Cell Sorting (FACS) to estimate the concentration (parasite per mL value). Based upon the estimated parasite per mL value, the sample was diluted in normal négative human whole blood to an estimated 6, 4, 2, 1, and 0.5 parasites per mL. The diluted Plasmodium-infected whole blood was lysed at a ratio of 0.9 mL of whole blood in 2.7mL of Parasite Transport Medium (PTM; 100 mM TRIS, 30 mM magnésium chloride, and 6% (v/v) LLS, at pH 7.5). Specimens were tested using TMA on the ftilly automated Procleix Panther system (Grifols Diagnostic Solutions Inc.). An assay calibrator comprising a P. falciparum IVT panel at 500 c/mL was included to détermine the analyte cutoff for the run. The assay software uses the analyte cutoff to détermine if samples are reactive or non-reactive. S amples with a signal to cutoff ratio of > 1 are considered reactive, while those < 1 are non-reactive. Assay reagents used included the following: a Target Capture Reagent (TCR) comprising TCOs of SEQ ID NO:9 and SEQ ID NO: 10; an Amplification reagent comprising T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol/rxn each); a Probe reagent comprising AE-labeled détection probes of SEQ ID NO: 148 and SEQ ID NO: 152 (1.9e6 RLU/rxn each); Ultrio Plus Enzyme reagent; and Sélection reagent.

Results are shown in Table 12 below. 95% LoD was 2.14 parasites/mL. 480 replicates each of internai control buffer, PTM, and négative lysate were evaluated with no false positives (0/1,440).

Table 12.

	50% LoD in Parasites/mL (Fiducial Limits)	95% LoD în Parasites/mL (Fiducial Limits)
P. falciparum	0.35 (0.14-0.56)	2.14(1.38-5.17)

Example 6: Interférence and Cross-reactivitv with Babesia

Interférence and cross-reactivity with Babesia microti (a homologous protozoan) was assessed using in vitro synthesized transcripts. Testing was performed in TMA reactions performed on the fully automated Procleix Panther System substantially as described in Example 2, with P. falciparum IVT dilutions between 10Ü-1 c/mL with and without the addition of B. microti IVT at le6 c/mL. The following oligomers were used: T7 oligomers of SEQ ID NO:71 and SEQ ID NO:72 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53 (5 pmol/rxn each), AE-labeled détection probes of SEQ ID NO: 148 and SEQ ID NO: 157 (1.9e6 RLU/rxn each), and TCOs of SEQ ID NO:9 and SEQ ID NO: 10.

Results are shown in Tables 13 and 14 below. Comparable rcactivîty and analyte RLU was observe for P. falciparum IVT with and without the presence of B. microti IVT.

Table 13.

	100	P. falciparum (copies)	0
30	10	3	1
% Reactive	Control	100	100	100	58	17	0
(N=24)	Presence of B. microti	100	100	96	58	17	0

Table 14.

	100	P. falciparum (copies)
30	10	3	1	0
Mcan RLU Control	2,043,991	2,031,427	1,993,527	1,992,755	2,001,796	2,518
(N-24) Presence of B. microti	2,055,344	2,029,613	1,925,711	1,777,978	1,815,085	855

Example 7: Evaluation of Redundant Probes

Redundant probe combinations were assessed using in vitro synthesized transcripts. Testing was performed in TMA reactions performed on the fully automated Procleix Panther System substantially as described in Example 2. The following oligomers were used: (i) T7 oligomers of SEQ ID NO:7I and SEQ ID NO:72 (5 pmol/rxn each), (ii) non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53 (5 pmol/rxn each), (iii) AE-labeled détection probes of SEQ ID NO: 148 (4,5 2MeAE linker) and SEQ ID NO: 157 (1.27e6 RLU/rxn each) with either SEQ ID NO: 148 (5,6 2MeAE linker) or SEQ ID NO: 148 (6,7 2MeAE linker), and (iv) TCOs of SEQ ID NO:9 and SEQ ID NO: 10.

Results are shown in Table 15 below, Cl refers to the probe combination containing SEQ ID NO: 148 (5,6 2MeAE linker); C2 refers to the probe combination containing SEQ ID 61

NO: 148 (6,7 2MeAE linker). Comparable reactivity was observer! for ail Plasmodium species at 30 and 10 c/mL with the redundant probe combinations. Consistent with results in Ex ample 1, probes having the same nucléotide sequence (SEQ ID NO: 148) but with different 2MeAE linker sites performed weil irrespective of their different labelling.

Table 15.

	P. faleiparum (copies)	P. knowlesi (copies)	P. malariae (copies)	P. ovule (copies)	P. vivax (copies)
30	lû	30	10	30	10	30	10	30	10
% Réactivé	Cl	100	88	100	100	100	100	100	75	100	100
(N=8)	C2	100	75	100	88	100	100	100	88	100	100

Example 8: Analvtical Sensitivitv - LoD of RNA Parasites/mL

Limit of détection (LoD) for a candidate amplification System was assessed by probit analysis using cultured P. falcipantm-infeci&l érythrocytes. Testing was performed in TMA reactions performed on the fully automated Procleix Panther System substantially as described in Example 5. The following oligomers were used: (i) T7 oligomers of SEQ ID NO:71 and SEQ ID NO:72 (5 pmol/rxn each), (ii) non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53 (5 pmol/rxn each), (iii) AE-labeled détection probes of SEQ ID NO:148 (4,5 2MeAE linker), SEQ ID NO:I57, and SEQ ID NO:I48 (5,6 2MeAE linker) ( 1.17e6, 7.92e5, and 2.03e6 RLU/rxn, respectively), and (iv) TCOs of SEQ ID NO:9 and SEQ ID NO: 10.

Results are shown in Table 16 below. 95% LoD was 2.31 parasites/mL. There were no false positives in the négative specimens tested. These results are comparable to the System evaluated in Example 5 where the 95% LoD was 2.14 parasites/mL.

Table 16.

	50% LoD în Parasites/mL (Fiducial Limits)	95% LoD in Parasites/mL (Fiducial Limits)
P. falciparutn	0.70 (0.33-0.97)	2.31 (1.61-5.82)

Example 9

Primer screening was performed using procedures for a manual Biphasic Real-Time TMA Assay. For the target capture step, 400 pL of Target Capture Reagent (TCR) comprising at least 1 Target Capture Oligo (TCO) and 1 T7 promoter provider was added to a 2mL Deep WelI 96-well plate (Thermo Scientific Cat. No. 95040450), followed by 500 pL of specimen. Specimens consisted of Plasmodium species in vitro transcript (IVT) diluted in buffer for

Plasmodium détection and Babesia species IVT diluted in buffet for Babesia détection. Specimens may also include B. microli IVT or P. falciparum IVT as a cross-reactivity specimen for opposing détection Systems due to the conserved régions between Babesia and Plasmodium. It is necessary to détermine that amplification and détection Systems are spécifie to the analyte System. The plate was covcred with a sealing card and loaded on to a Torrcy Pines plate incubator and covered with the lîd. Incubation steps for the Torrey Pines incubator included 7 minutes at 80°C followed by 17 minutes at 62°C and between 15 to 25 minutes at 25°C, respect! vel y.

After target capture incubation steps, the plate was loaded with a deep well comb tip (Thermo Scientific Cat. No. 97002534) and placed on to a Kingfisher 96 instrument (Thermo Scientific Type 710 R EF 5400500) fitted with deep well magnets. The Kingfisher instrument was additionally loaded with a wash plate consisting of a 2mL 96-weIl plate (Nunc Deep Well plate Cat. No. 278752) prepared with 500 pL of commercially available Procleix Wash Buffer reagent (Grifols Diagnostic Solutions Inc.) and a second wash plate consisting of a 200μL 96well plate (Thermo Scientific Cat. No. 97002540) containing 200 pL of the wash buffer. For the deep well wash steps, the plate containing the hybridîzed TCR-sample mixture was mixed for 5 minutes before collecting magnetic beads for 20 counts and eluting for 20 seconds to the plate containing 500 pL of wash buffer. The 500 pL wash plate was mixed for 1 minute before collecting magnetic beads for 10 counts and eluting for 20 seconds to the wash plate containing 200 pL of wash buffer.

The second wash plate containing the mixture of hybridîzed magnetic beads and wash buffer was removed from the Kingfisher instrument, loaded with a small PCR tip comb (Thermo Scientific 97002514) and transferred to a second Kingfisher 96 instrument (Thermo Scientific Type 710 REF 5400500) fitted with PCR magnets. The Kingfisher instrument was additionally loaded with a 96-well PCR plate (Axygen Cat. No. PCR-96-HS-C) containing 30 pL of Amplification Reagent (without Phénol Red) containing at least 1 non-T7 primer (amplification plate). To transfer the hybridîzed magnetic beads to the amplification plate the wash plate was mixed for 5 minutes before collecting magnetic beads for 30 counts and eluting for 30 seconds to the amplification plate. The wash plate was mixed again for 1 minute before collecting magnetic beads for 30 counts and eluting for 30 seconds to the amplification plate to complété the transfer.

The amplification plate was covered with a sealing card and loaded on to a Stratagene instrument (Mx3005P Multiplex Quantitative PCR System) to incubate for 5 minutes at 43 °C. The plate was transferred to a heat block set to 42°C and uncovered to add 10 pL of commercially available Ultrio Plus Enzyme reagent (Grifols Diagnostic Solutions Inc.) and re covered with a sealing card. The plate was mixed on the heat block for 1 minute at 1400 RMP and reloaded on the Stratagene instrument to incubate for 5 minutes at 43°C. The plate was transferred again to the heat block and uncovered to add 15 pL of Promoter reagent (Amplification Reagent without Phénol Red) containing a mixture of at least 1 T7 promoter provider and at least 1 Fluorescent labeled molecular Torch or Beacon (5’-HexochloroFluorescein (HEX) for Plasmodium or 5’-Fluorescein (FAM) for Babesia) and sealed with a clear adhesive plate cover, The plate was mixed on the heat block for l minute at 1400 RMP and reloaded on the Stratagene for the read protocol. The read protocol consisted of incubation at 43°C and read every 30 seconds for cycles of 120 or 150.

The raw data exported from the Stratagene instrument was analyzed using an in-house software tool. Fluorescent curves were analyzed using a threshold of 1,000 Relative Fluorescent Units (RFU). The time for specîmens to meet or exceed the threshold (TTime) was detennined by the software. Specîmens with a TTime were considered Reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. Lower TTimes indicated better performance of the tested Systems. Specîmens with no TTime, or under the threshold, were considered non-reactive.

In this ex ample, combinations of molecular torch or beacon probes and amplification oligomer pairs were tested in three groups (Groups 1, 2, and 3). Tested oligomer combinations are shown in Table 17 below.

Table 17. Combinations of Amplification Oligomers and Torch Probes.

T7 Primer	Non-T7 Primer	Probe (Torch/Beacon)	Group
SEQ ID NO:181	SEQ ID NO:183	SEQ ID NO: 104 (torch)	Group 1
SEQ ID NO:181	SEQ ID NO:183	SEQ ID NO: 105 (torch)
SEQ ID NO:181	SEQ ID NO:183	SEQ ID NO: 106 (torch)
SEQ ID NO:181	SEQ ID NO: 183	SEQ ID NO:121 (torch)
SEQ ID NO: 181	SEQ IDNO:183	SEQ ID NO: 107 (beacon)
SEQ ID NO:181	SEQ ID NO:183	SEQ ID NO: 108 (beacon)
SEQ ID NO:181	SEQ ID NO:184	SEQ ID NO: 104 (torch)	Group 2
SEQ 1DNO:59	SEQ IDNO:51	SEQ ID NO:123 (torch)	Group 3
SEQ ID NO:59	SEQ ID NO:31	SEQ ID NO:123 (torch)
SEQ ID NO:59	SEQ ID NO:50	SEQ ID NO:123 (torch)

Group 1 Results. Probes of SEQ ID NOs: 104-107 and 121 successftilly detected

Plasmodium with fluorescent curves generating average TTimes of less than or equal to 30.69 minutes at 10 c/mL. No fluorescent curves with TTimes were generated for the probe of SEQ ID NO: 108 concluding it was unable to detect Plasmodium.

Group 2 Results. Amplification oligomers of SEQ ID NOs:181 and 184 in combination with the torch probe of SEQ ID NO: 104 successfully detected Plasmodium, demonstrated by fluorescent curves generating TTÎmes of less than or cqual to 20.89 minutes at 10 c/mL, and showed no cross-reactivity with Babesia 1VT, demonstrated by the absence of a fluorescent curve with a TTime.

Group 2 Results. Non-T7 amplification oligomers of SEQ ID NOs:51, 31, and 50 each in combination with the T7 amplification oligomer of SEQ ID NO:59 and the torch probe of SEQ ID NO: 123 successfully detected Plasmodium as demonstrated by fluorescent curves generating TTimes of less than or equal to 22.82 minutes.

Example 10

A candidate amplification System was tested using Real-time TMA on the fully automated Panther System installed with Real-time Fluorometers (Hologic Inc.). Specimens consîsted of Plasmodium species IVT diluted in buffer for Plasmodium détection and Babesia species IVT diluted in buffer for Babesia détection. Specimens may also include B. microti IVT or P. falciparum IVT as a cross reactivity specimen. It is necessary to détermine that amplification and détection Systems are spécifie to the analyte System. Assay Reagents used included the following: a TCR comprising of at least one TCO and one T7 promoter provider; an Amplification Reagent comprising of at least one non-T7 primer; a Promoter reagent (Amplification Reagent without Phénol Red) consisting of at least one T7 promoter provider and ai least one Fluorescent labeled molecular Torch or Beacon (5’-Hexochloro-Fluorescein (HEX) for Plasmodium or 5’-Fluorescein (FAM) for Babesia)', and Ultrio Plus Enzyme reagent.

For analysis of the raw data exported from the Panther System, the Panther RT-Dev Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of 1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the threshold (TTime) was determined by the software. Specimens with a TTime were considered Reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. Lower TTimes indicated better performance of the tested Systems. Specimens with no TTime, or under the threshold, were considered nonreactive.

The oligomers tested in this ex ample were a T7 oligomer of SEQ ID NO:59, a non-T7 oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO: 123. Results demonstrated détection of 8 out of 8 replicates tested at 30 c/mL of Plasmodium IVT and 3 out of 8 replicates tested at 10 c/mL. Fluorescent curves generated TTimes of less than or equal to 33.18 minutes for the detected replicates at 10 c/mL.

Example 11: Détection of Plasmodium in Clinical Specimens

Candidate amplification Systems were tested using Real-time TMA on the fully automated Panther system installed with Real-time Fluorometers (Hologic Inc.). Specimens consisted of positive Controls for B. microti and P. falciparum IVT diluted in buffer to 300 c/mL and a négative control consisting of négative buffer. Clinical specimens consisted of P. falciparum and P. ovale whole blood and plasma specimens. Whole blood specimens were prepared by manual addition of 100 pL to 3 mL of a lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v/v) LLS, and 0.1 mM EDTA, at a pH of 7.4). Plasma specimens were prepared by manual addition of 100 pL to 3 mL of processed human plasma. Specimens were tested on the Panther system. Assay Reagents used included the following: a TCR comprising of at least one TCO and one T7 promoter provider; an Amplification Reagent comprising of at least one non-T7 primer; a Promoter reagent (Amplification Reagent without Phénol Red) consisting of at least one T7 promoter provider and at least one Fluorescent labeled molecular Torch or Beacon (5'-HexochIoro-Fluorescein (HEX) for Plasmodium or 5-Fluorescein (FAM) for Babesiap, and Enzyme reagent.

For analysis of the raw data exported from the Panther system, the Panther RT-Dev Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of 1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the threshold (TTime) was determined by the software. Specimens with a TTime were considered Reactive for Plasmodium in the FIEX channel or Babesia in the FAM channel. Lower TTimes indicated better performance of the tested Systems. Specimens with no TTime, or under the threshold, were considered Nonreactive.

The oligomers tested in this example were a T7 oligomer of SEQ ID NO:59, a non-T7 oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO: 123. Results are shown in Table 18 below. Real-time TMA results with the tested oligomers showed 100% concordance with PCR results. Based on the calibration curve generated using in vitro Plasmodium falciparum infected érythrocytes, the tested sample hâve between 7.14E6 and 2.14E8 parasites/mL in whole blood. No cross reactivity was observed with Babesia.

Table 18. Détection of Plasmodium positive Clinical Specimens.

		Whole Blood	Plasma	Whole Blood	Plasma
Sample	Organism	PCR Result (Wadsworth)	Real-time TMA #reactive/ #tested	Real-time TMA #reactive/ #tested	Est.# par/mL	Est.# par/mL	Relative aniouiits of parasites (WB/pfasma)
l	P-fal	Positive	4/4	4/4	2.14E+08	3.18E+02	672,841
2	P. fai	Positive	4/4	4/4	1.49E+08	2.63E+02	566,724
3	P.fal	Positive	4/4	4/4	1.11E+08	2.55E+03	43,685
4	P.fal	Positive	4/4	4/4	7.28E+07	4.60E+02	158,340
5	P-fal	Positive	4/4	4/4	5.49E+07	1.08E+02	508,948
6	P.fal	Positive	4/4	4/4	3.07E+07	1.96E+01	1,568,437
7	P.fal	Positive	4/4	4/4	2.27E+07	2.22E+01	1,026,604
8	P-fal	Positive	4/4	4/4	1.09E+07	2.38E+01	457,997
9	P. ova	Positive	4/4	4/4	1.05E+07	3.21E+04	329
10	P-fal	Positive	4/4	4/4	7.14E+06	1.74E+01	411,025

Example 12

Candidate amplification Systems were tested using Real-time TMA on the fully automated Panther System installed with Real-time Fluorometers (Hologic Inc.). Specimens consisted of 5 strains of Plasmodium infected érythrocytes: US 05 F Bénin I, US 05 F Santa Lucia, US 08 F Nigeria XII, US05 F FC27/A3, and US 05 F PHI. Infected érythrocytes were provided with an estimated parasite/mL value. Each strain was serially diluted in normal négative human whole blood to estimated values of 10 parasîtes/mL. The diluted Plasmodiuminfected whole blood was manually lysed at a ratio of 1 mL of whole blood in 3 mL of a lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v/v) LLS, and 0.1 mM EDTA, at a pH of 7.4). Specimens were tested on the Panther System. Assay Reagents used inctuded the following: a TCR comprising of at least one TCO and one T7 promoter provider; an Amplification Reagent comprising of at least one non-T7 primer; a Promoter reagent (Amplification Reagent without Phénol Red) consisting of at least one T7 promoter provider and at least one Fluorescent labeied molecular Torch or Beacon (5’-Hexochloro-FIuorescein (HEX) for Plasmodium or 5’-Fluorescein (FAM) for Babesia)', and Enzyme reagent.

For analysis of the raw data exported from the Panther System, the Panther RT-Dev Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of 1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the threshold (TTime) was determined by the software. Specimens with a TTime were considered Reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. Lower TTimes indicated better perfonnance of the tested Systems. Specimens with no TTime, or under the threshold, were considered

Nonreactive.

The oligomers tested in this ex amp le were a T7 oligomer of SEQ ID NO: 59, a non-T7 oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO: 123. Results dcmonstrated that ail 5 5 strains of Plasmodium infected érythrocytes were detected in 6 out of 6 replicates tested at 10 parasites/mL with fluorescent curves generating TTimes ranging from 14.29 to 17.17 minutes.

Table 19. Exemplary Sequences.

SEQ ID NO:	Sequence (5’ to 3')	Commenta
1	GGAUUGGGUAAUUUGCGCGCCCTTTAAAAAAAAAAAAAAA aaaaaaaaaaaaaaa	Target capture oligomer
2	CAAGAAAGAGCUAUCAAUCUGUCAAUCCTTTAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA	Target capture oligomer
3	CCCGUGUUGAGUCAAAUUAAGCCGCATTTAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA	Target capture oligomer
4	GGGUAAUUUGCGCGCCUGCUGCTTTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA	Target capture oligomer
5	UUUCUCAGGCUCCCUCUCCGGAAUCGTTTAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA	Target capture oligomer
6	ACAUCUGAAUACGAAUGUCCCCAATTTAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA	Target capture oligomer
7	CUAGUCGGCAUAGUUUAUGGUUATTTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA	Target capture oligomer
8	AAAAACGGCCAUGCAUCACCAUCCTTTAAAAAAAAAAAAA ΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑ	Target capture oligomer
9	UAGGCCAAUACCCUACCGUCCTTTAAAAAAAAAAAAAAAA AAAAAAAAAAAAAA	Target capture oligomer
10	AAAGACUUUGAUUUCUCGCAAGGTTTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA	Target capture oligomer
11	GGAUUGGGUAAUUUGCGCGCCC	THS ofSEQIDNO:l
12	CAAGAAAGAGC UAUCAAUC UGUCAAUCC	THS ofSEQ 1DNO:2
13	CCCGUGUUGAGUCAAAUUAAGCCGCA	THS ofSEQIDNO:3
14	GGGUAAUUUGCGCGCCUGCUGC	THS of SEQIDNO:4
15	UUUCUCAGGCUCCCUCUCCGGAAUCG	THS ofSEQ ID NO:5
16	ACAUC UGAAUACGAAUG UCCCCAA	THS ofSEQIDNO:6
17	CUAGUCGGCAUAGUUÜAUGGUUA	THS of SEQ ID NO:7
18	AAAAACGGCCAUGCAUCACCAUCC	THS of SEQIDNO:8
19	UAGGCCAAUACCCUACCGUCC	THS of SEQIDNO:9
20	AAAGACUUUGAUUUCUCUCAAGG	THS ofSEQ IDNO:10
21	AATAC TACAGCATGG	Non-T7 amp oligo

22	GGAAGGCAGCAGGCGCGTA	Non-T7 amp oligo
23	AATACTACAGCATGGA	Non-T7 amp oligo
24	AATACTACAGCATGGAA	Non-T7 amp oligo
25	ATACTACAGCATGGAATA	Non-T7 amp oligo
26	ATTCAGATGTCAGAGGTGA	Non-T7 amp oligo
27	GTATTCAGATGTCAGAGGTGA	Non-T7 amp oligo
28	GTTACGATTAATAGGAGT	Non-T7 amp oligo
29	GTTACGATTAATAGGAGTA	Non-T7 amp oligo
30	GTTACGATTAATAGGAGTAG	Non-T7 amp oligo
31	G T TAC GATTAATAG GAG TAGC	Non-T7 amp oligo
32	AATAC TACAGCAT G GAAT	Non-T7 amp oligo
33	AATAC TACAGCAT GGAATA	Non-T7 amp oligo
34	TACGAT TAATAGGAGT	Non-T7 amp oligo
35	TACTACAGCATGGAATA	Non-T7 amp oligo
36	TAT T CAGAT GT CAGAGGT GA	Non-T7 amp oligo
37	TCAGTNCCTTATGAGAAATC	Non-T7 amp oligo
38	TGGCTTAGTTACGATT	Non-T7 amp oligo
39	TGGCTTAGTTACGATTAATAG	Non-T7 amp oligo
40	TTAATAGGAGTAGCTTGGGG	Non-T7 amp oligo
41	TTACGATTAATAGGAGT	Non~T7 amp oligo
42	TT CAGATGT CAGAGGTGA	Non-T7 amp oligo
43	TTGGCTTAGTTACGAT	Non-T7 amp oligo
44	TTGGCTTAGTTACGATTA	Non-T7 amp oligo
45	TTGGGGACATTCGTATTCAGA	Non-T7 amp oligo
46	TTTAGATTGCTTCCTTCAGT	Non-T7 amp oligo
47	TTTGAATACTANAGCA	Non-T7 amp oligo
48	ACATTCGTATTCAGATGTCAG	Non-T7 amp oligo
49	CTTAGTTACGATTAATAGGA	Non-T7 amp oligo
50	CGATTAATAGGAGTAGCTTGG	Non-T7 amp oligo
51	C TTAGTTACGAT TAATAGGAGTAG	Non-T7 amp oligo
52	C T T GAATACT NCAG CA	Non-T7 amp oligo
53	GGCTTAGTTACGATTA	Non-T7 amp oligo
54	AATACTANAGCATGG	Non-T7 amp oligo
55	AATAC TANAGCAT GGAATA	Non-T7 amp oligo
56	AATT C TAAAGAAGAGAG	Non-T7 amp oligo
57	AATT TAATACGACTCACTATAGGGAGATT CACT CC CT TAA CTTTCGTTCTTG	T7 amp oligo
58	AATTTAATACGACTCACTATAGGGAGACTTGATTAATGGA	T7 amp oligo

	AGTATTTTAGA
59	AATTTAATACGACTCACTATAGGGAGACTT AACTTTCGTT CTTGATTAATGGAAGT	T7 amp oligo
60	AATTTAATACGACTCACTATAGGGAGACCTACTCTTGTCT TAAACTA	T7 amp oligo
61	AATTTAATACGACTCACTATAGGGAGAAAACGGCCATGCA TCACCATCCAAGA	T7 amp oligo
62	AATTTAATACGACTCACTATAGGGAGACTCCCTTAACTTT CGTTCTTGATTAATGGAAGT	T7 amp oligo
63	AATTT.AATACGACTCACTATAGGGAGACGACGGTATCTGA TCGTCTTCACTCCC	T7 amp oligo
64	AAT TTAATAC GACT CAC TATAGGGAGACTTAAC T TT CGT T C T T GATTAAT GGAAG	T7 amp oligo
65	AATTTAATACGACTCACTATAGGGAGACTTAACTTTCGTT CTTGATTAATGGAAGTA	T7 amp oligo
66	AATTTAATACGACTCACTATAGGGAGACACTCCCTTAACT TTCGTTCTTGATTAATG	T7 amp oligo
67	AATTTAATACGACTCACTATAGGGAGACACTCCCTTAACT TTCGTTCTTGATTAATGG	T7 amp oligo
68	AATTTAATACGACTCACTATAGGGAGACTTCACTCCCTTA ACTTTCGTTCTTGATT	T7 amp oligo
69	AAT T TAATACGAC T CACTATAGGGAGACT T CAC T CCC T TA ACTTTCGTTCTTGAT	T7 amp oligo
70	AAT T TAATAC GACT CAC TATAGGGAGAATC GT C T TCACTC CCTTAACTTTCGTTC	T7 amp oligo
71	AATTTAATACGACTCACTATAGGGAGACTCCCTTAACTTT CGTTCTTGATTAATG	T7 amp oligo
72	AATTTAATACGACTCACTATAGGGAGATCACTCCCTTAAC TTTCGTTCTTGAT	T7 amp oligo
73	AATTTAATACGACTCACTATAGGGAGACCCTTAACTTTCG TTCTTGATTAATG	T7 amp oligo
74	AATT TAATACGAC TCACTATAGGGAGACT TAACT T T C GT T CTTGATTAATG	T7 amp oligo
75	AATT TAATACGAC T CACTATAGGGAGATAAC TTTCGTTCT TGATTAATG	T7 amp oligo
76	AAT TTAATACGAC T CAC TATAGGGAGAAC T CCC T TAACT T TCGTTCTTGAT	T7 amp oligo
77	AAT T TAATACGACT CAC TATAGGGAGAT CC CT TAAC TT TC GTTCTTGAT	T7 amp oligo
78	AAT TT AATACGACT CAC TATAGGGAGAAGGCAAAT GCT TT CGCAGTTGTTNGTCT	T7 amp oligo
79	AAT TTAATAC GACTCAC TATAGGGAGAAGGCAAAT GCT TT CGCAGTTGTTTGTCT	T7 amp oligo
80	TTCACTCCCTTAACTTTCGTTCTTG	THS ofSEQIDNO:57
81	C T TGATT AATGGAAGTAT TT TAGA	THS ofSEQIDNO:58
82	CTTAACTTTCGTTCTTGATTAATGGAAGT	THS ofSEQIDNO:59
83	CCTACTCTTGTCTTAAACTA	THS ofSEQIDNO:60
84	AAACGGCCATGCATCACCATCCAAGA	THS of SEQ CD NO:61

85	CTCCCTTAACTTTCGTTCTTGATTAATGGAAGT	THS of SEQ ID NO:62
86	CGACGGTATCTGATCGTCTTCACTCCC	THS ofSEQ ID NO:63
87	CTTAACTTTCGTTCTTGATTAATGGAAG	THS of SEQIDNO:64
88	CTTAACTTTCGTTCTTGATTAATGGAAGTA	THS ofSEQ ID NO:65
89	CACTCCCTTAACTTTCGTTCTTGATTA4TG	THS ofSEQ ID NO:66
90	CACTCCCTTAACTTTCGTTCTTGATTAATGG	THS ofSEQ IDNO:67
91	CTTCACTCCCTTAACTTTCGTTCTTGATT	THS ofSEQ ID NO:68
92	CTTCACTCCCTTAACTTTCGTTCTTGAT	THS ofSEQ ID NO:69
93	ATCGTCTTCACTCCCTTAACTTTCGTTC	THS of SEQ ID NO:70
94	CTCCCTTAACTTTCGTTCTTGATTAATG	THS ofSEQ IDNO:71
95	TCACTCCCTTAACTTTCGTTCTTGAT	THS ofSEQ ID NO:72
96	CCCTTAACTTTCGTTCTTGATTAATG	THS ofSEQ IDNO:73
97	CTTAACTTTCGTTCTTGATTAATG	THS ofSEQ ID NO:74
98	TAACTTTCGTTCTTGATTAATG	THS of SEQ ID NO:75
99	ACTCCCTTAACTTTCGTTCTTGAT	THS ofSEQ ID NO:76
100	TCCCTTAACTTTCGTTCTTGAT	THS ofSEQ ID NO:77
101	AGGCAAATGCTTTCGCAGTTGTTNGTCT	THS ofSEQ ID NO;78
102	AGGCAAATGCTTTCGCAGTTGTTTGTCT	THS ofSEQ ID NO:79
103	CGCGCAAGCGAGAAAGCGCG	Torch détection probe
104	GCUCGCAUUCGCGCAAGCGAGC	Torch détection probe
105	GCUUGCGAGUAUUCGCGCAAGC	Torch détection probe
106	GGCAAGCGAGAAAGUCUUGCC	Torch détection probe
107	CCGAGGUAUUCGCGCAACUCGG	Beacon détection probe
108	GGCUCACUUUCUCGCUUGGAGCC	Beacon détection probe
109	C UC UGGAGACNAG CACCAGAG	Torch détection probe
110	G UC UCAU U UUC UG GAGAC	Torch détection probe
111	GCCUAAAAUACUUCCUAGGC	Torch détection probe
112	CAUGGAAAUACUUCCAUG	Torch détection probe
113	C CGAGAUUUUCUGGAGACC UC GG	Beacon détection probe
114	CCGAGGCCUAAAAUACUUCCCUCGG	Beacon détection probe
115	GGGAAUUUAAAACCUUCCC	Torch détection probe
116	GGAAGGAAUUUAAAACCUUCC	Torch détection probe
117	GUGGGAAUUUAAAACCCCCAC	Torch détection probe
118	UCCAGAAAUUCUUAGAUUUUCÜGGA	Torch détection probe
119	ACUCCGAACGAAAGUUAAGGGAGU	Torch détection probe
120	AGGGAGUGAAGACGAUCAUCCCU	Torch détection probe
121	UCGCGCAAGCGAGAAAGGCGCGA	Torch détection probe
122	CCGAAGUGNCUAAAAUACUUCGG	Torch détection probe

123	CACCUCAGAUGUCAGAGGUG	Torch détection probe
124	C UACC UC UAAAGAAGAGAGG UAG	Torch détection probe
125	CGCGCAAGCGAGAAAG	THS ofSEQ ID NO:103
126	AUUCGCGCAAGCGAGC	THS ofSEQ ID NO: 104
127	GAGÜAUUCGCGCAAGC	THS ofSEQ ID NO: 105
128	GGCAAGCGAGAAAGU	THS ofSEQ ID NO:106
129	GUAUUCGCGCAA	THS ofSEQ ID NO:107
130	ACUUUCUCGCUUG	THS ofSEQ ID NO:108
131	CUCUGGAGACNAGCA	THS ofSEQ ID NO: 109
132	AUUUUCUGGAGAC	THS ofSEQ ID NO: 110
133	GCCUAAAAUACUUCC	THS ofSEQ IDNO:111
134	AAAUACUUCCAUG	THS ofSEQ IDNO:112
135	AUUUUCUGGAGAC	THS of SEQ ID NO: 113
136	CCUAAAAUACUUC	THS ofSEQ ID NO: 114
137	GGGAAUUUAAAACC	THS ofSEQ ID NO:115
138	GGAAUUUAAAACCUUCC	THS ofSEQ ID NO: 116
139	GUGGGAAUUUAAAACC	THS ofSEQ ID NO: 117
140	GAAAUUCUUAGAUUUUCUGGA	THS ofSEQ IDNO:118
141	GAAC GAAAG UUAAGGGAGU	THS ofSEQ ID NO: 119
142	AGGGAGUGAAGACGAUCA	THS ofSEQ ID NO: 120
143	UCGCGCAAGCGAGAAAG	THS ofSEQ ID NO: 121
144	UGNCUAAAAUACUUCGG	THS ofSEQ ID NO:122
145	CAGAUGUCAGAGGUG	THS of SEQ ID NO: 123
146	UCUAAAGAAGAGAGGUAG	THS of SEQ ID NO: 124
147	UC U UAGAUUUUC UGGAGAC	Détection probe
148	UUCAGAUGUCAGAGG	Détection probe
149	UUCAGAUGUCAGAGGT	Détection probe
150	UAUUCAGAUGUCAGAGGT	Détection probe
151	UAUUCAGAUGUCAGAGGUG	Détection probe
152	UCAGAUGUCAGAGGT	Détection probe
153	UUCAGAUGUCAGAGGT	Détection probe
154	AUUCAGAUGUCAGAGGT	Détection probe
155	AUUCAGAUGUCAGAGGUG	Détection probe
156	CUUAGAUUUUCUGGAGA	Détection probe
157	CUUAGAUUU UC UGGAGAC	Détection probe
158	CUUAGUUACGAUUAAUAGGA	Détection probe
159	GUAUUCAGAUGUCAGAGGUGA	Détection probe
160	AUÜCUUAGAUUUUCUGGAGAC	Détection probe

161	CUAAGAUUUUCUGGAGAC	Détection probe
162	TTTGAATACTANAGCATGGAATA	Amp oligo hybridîzing région (SEQ ID NOs:21, 23-25, 32, 33, 35, 54, & 55 are contained within here)
163	TACTANAGCA	Amp oligo core sequence (SEQ ID NOs:21/23-25, 32, 33, 35, 54, & 55 share this)
164	TTTGAATACTACAGCATGGAATA	Amp oligo hybridîzing région (SEQ ID NOs:21,23-25, 32, 33, & 35 are contained within here)
165	TACTACAGCA	Amp oligo core sequence (SEQ ID NOs:21, 23-25, 32, 33, & 35 share this)
166	TTGGCTTAGTTACGATTAATAGGAGTAGCTTGGGG	Amp oligo hybridîzing région (SEQ ID NOs:28-31, 34, 38-41, 43, 44, 49-51, & 53 are contained within here)
167	TTAATAGGAGT	Amp oligo core sequence (SEQ ID NOs:28-31, 34, 40, 41, & 49-51 share this)
168	GGCTTAGTTACGAT	Amp oligo core sequence (SEQ ID NOs:38, 39, 43, 44, & 53 share this)
169	CGACGGTATCTGATCGTCTTCACTCCCTTAACTTTCGTTC T TGAT TAATGGAAGTAT TT TAGA	Amp oligo hybridîzing région (SEQ ID NOs :80-82, & 85-100 are contained within here)
170	ATCGTCTTCACTCCCTTAACTTTCGTTCTTGATTAATGGA AGTATTTTAGA	Amp oligo hybridîzing région (SEQ ID NOs:80-82, 85, & 87-100 are contained within here)
171	CTTGATTAATG	Amp oligo core sequence (SEQ ID NOs:81, 82, 85, 87-90, 94, & 96-98 share this)
172	TAACTTTCGTTC	Amp oligo core sequence (SEQ ID NOs:8Û, 82, 85, & 87-100 share this)
173	CTTCACTCCC	Amp oligo core sequence (SEQ ID NOs:86 & 91-93 share this)
174	UCAGAUGUCAGAGG	Détection probe core sequence (SEQ ID NOs:148-155 & 159 share this)
175	C UAAGAUU UUCUGGAGA	Détection probe core sequence (SEQ ID NOs:147, 156, 157, 160, & 161 share this)
176	CAGAUGUCAGAGG	Détection probe core sequence (SEQ ID NOs: 145, 148-155, & 159 share this)
177	YUCUGGAGAC	Détection probe core sequence (SEQ ID NOs:131, 132, 135, 147, 156, 157, 160, & 161 share this)
178	AUUUUCUGGA	Détection probe core sequence (SEQ ID NOs: 132, 135, 140, 147, 156, 157, 160, & 161 share this)
179	AAT TTAATAC GACT CAC TATAGGGAGA	Exemplary T7 promoter sequence for use in an isothermal amplification

		reaction
180	aacctggttgatcttgccagtagtcatatgcttgtctcaa agaLtaagccatgcaagtgaaagtatataratatüttata tgtagaaactgcgaacggctcattaaaacagttatagtct acttgacatttttattataaggataactacggaaaagctg tagctaatacttgctttattatcctttgatttttatcttt ggataagtatt tgt caggect n àtaagaaaaaagttatta acttaaggaattataacaaagaagtaacacgtaataaatt tactc ta t ttagtgtgtatcaat cgagtt tctgaccta te agett ttgatgt tagggtat tggcctaacatggctatgac gggtaacggggaattagagttcgattccggagagggagcc tgagaaatagctaccacatctaaggaaggaagcaggcgcg taaattacccaattctaaagaagagaggtagtgacaagaa ataacaatgcaaggccaatttttggttt tgtaattggaat ggtgggaatttaaaacettcccagagtaacaattggaggg caagtctggtgccagcagccgcggtaattccagctccaat agcgtatattaaaattgttgcagttaaaacgctcgtagtt gaatttcaaagaatcgatattttattgtaactat tetagg ggaactattttagctttcgctttaatacgcttcctctatt attatgttctttaaataacaaagattctttttaaaatccc cacttttgcttttgctttttggggattttgttactttgag taaattagagtgttcaaagcaaacagttaaagcatttact gtgtttgaatactatagcatggaataacaaaattgaacaa gctaaaattttttgttcttttttcttattttggcttagtt acgattaataggagtagettggggacattcgtattcagat gtcagaggtgaaattcttagattttctggagacgaacaac tgcgaaagcatttgtctaaaatacttccattaatcaagaa cgaaagttaagggagtgaagacgatcagataccgtcgtaa tcttaaccataaactatgccgactaggtgttggatgaaag tgttaaaaataaaagtcatctttcgaggtgacttttagat tgcttccttcagtaccttatgagaaatcaaagtctttggg ttctggggcgagtattcgcgcaagcgagaaagttaaaaga attgacggaagggcaccaccaggcgtggagcttgcggctt aatttgactcaacacggggaaactcactagtttaagacaa gagtaggattgacagattaatagctctttcttgatttctt ggatggtgatgcatggccgtttttagttcgtgaatatgat ttgtctggttaattccgataacgaacgagatcttaacctg ctaattagcggcgagtacactatattcttatttgaaattg aacataggtaactatacatttattcagtaatcaaattagg atatttttattaaaatatccttttccctgttctactaata atttgttttttactctatttctctcttcttttaagaatgt acttgcttgattgaaaagcttcttagaggaacattgtgtg tctaacacaaggaagtttaaggcaacaacaggtctgtgat gtccttagatgaactaggctgcacgcgtgctacactgata tatataacgagtttttaaaaatatgcttatatttgtatct ttgcttatattttgcatacttttcctccgccgaaaggcgt aggtaatctttatcaatatatatcgtgatggggatagatt attgeaattattaatcttgaacgaggaatgcctagtaagc atgattcatcagattgtgctgactacgtccctgccctttg tacacaccgcccgtcgctcctaccgattgaaagatatgat gaattgtttggacaagaaaaattgaattatattctttttt tttctggaaaaaccgtaaatcctatcttttaaaggaagga gaagtcgtaacaaggtttccgtaggtgaacctgcggaagg atcatta	Plasmodium falciparum 3D7 I8S ribosomal RNA (PF3D7_0725600), rRNA NCBI Référencé Sequence; XR_002273081.2
181	ΑΆΤ TTAATAC GACT CACTATAGGGAGAT CAAGAAAG AGCT ATNAATCTGTCAATCC	T7 amp olîgo
182	TCAAGAAAGAGC TATNAAT CT GT CAATC C	THS of SEQ ID NO:181
183	GAAATCAAAGTCTTTGGGTTCTG	Non-T7 amp olîgo
184	CAAAGTCTTTGGGTTCTGG	Non-T7 amp olîgo
185	TTTAGATTGCTTCCTTCAGTNCCTTATGAGAAATCAAAGT CTTTGGGTTCTGG	Amp olîgo hybridizing région (SEQ ID

		NOs:37, 46, 183, & 184 are contained within here)
186	GAAATCAAAGTCTTTGGGTTCTGG	Amp oligo hybridizing région (SEQ ID NOs: 183 & 184 are contained within here)
187	CAAAGTCTTTGGGTTCTG	Amp oligo core sequence (SEQ ID NOs: 183 & 184 share this)
188	AAACGGCCA.TGCATCACCATCCAAGAAATCAAGAAAGAGC TATNAATCTG'TCAATCCTACTCTTGTCTTAAACTA	Amp oligo hybridizing région (SEQ ID NOs:83, 84, & 182 are contained within here)
189	GAGUAU OC G S GCAAG CGAGAAAG□	Détection probe hybridizing région (SEQ ID NOs: 125-130 & 143 are contained within here)
190	AUUCGCGCAA	Détection probe core sequence (SEQ ID NOs;126, 127, & 129 share this)
191	CAAGCGAGC	Détection probe core sequence (SEQ ID NOs:125, 126, 128, 130, & 143 share this)
192	[GenBank Accession JQ627153.1]	Plasmodium vivax isolate SV 1 18S ribosoma] RNA gene, partial sequence
193	[GenBank Accession L0756Û.1]	Plasmodium knowlesi small subunit ribosomal RNA sequence
194	[GenBank Accession AB182491.1J	Plasmodium ovale gene for small subunit ribosomal RNA, complété sequence, variant type 1
195	[GenBank Accession AF487999.1]	Plasmodium cf, malariae type 1 small subunit ribosomal RNA gene, complété sequence
196	GUAUUCAGAUGUCAGAGGUGAAAUUCUUAGAUUYUCUGGA GACNAGCA	Détection probe hybridizing région (SEQ ID NOs:131, 132, 135, 140, 145, 147-157 & 159-161 are contained within here)
197	GUAUUCAGAUGUCAGAGGUGAAAÛUCUUAGAUUUUCUGGA GAC	Détection probe hybridizing région (SEQ ID NOs:132, 135, 140, 145, 147157 & 159-161 are contained within here)

EMBODIMENTS

Embodiment 1. A method for specifically detecting Plasmodium species nucleic acid in a sample, said method comprising:

(1) contacting a sample, said sample suspected of containing Plasmodium species nucleic acid, with at least two oligomers for amplifying a target région of a Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:

(a) an amplification oligomer comprising a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163; or (ii) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and comprises the sequence ofSEQ ID NO: 167 or SEQ ID NO: 168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO:171, SEQ ID NO: 172, or SEQ ID NO;173;

(2) performing an in vitro nucleic acid amplification reaction, wherein any Plasmodium target nucleic acid présent in said sample is used as a tempiate for generating an amplification product; and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence ai Plasmodium species target nucleic acid in said sample.

Embodiment 2. The method of Embodiment I, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(i).

Embodiments. The method of Embodiment 2, wherein the target-hybridizing sequence of (a)(i) is selected from the group consisting of SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55.

Embodiment 4. The method of Embodiment 2, wherein the target-hybridizing sequence of (a)(i) is contained in the sequence of SEQ ID NO: 164 and comprises the sequence ofSEQ ID NO: 165.

Embodiment 5. The method of Embodiment 4, wherein the target-hybridizing sequence of (a)(i) is selected from the group consisting ofSEQ ID NOs:21, 23-25, 32, 33, and 35.

Embodiment 6. The method of Embodiment 1, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(ii).

Embodiment 7. The method of Embodiment 6, wherein the target-hybridizing sequence of (a)(ii) comprises the sequence ofSEQ ID NO: 167.

Embodiment 8. The method of Embodiment 7, wherein the target-hybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs:28-31, 34, 40, 41, and 49-51.

Embodiment 9. The method of Embodiment 6, wherein the target-hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 168.

Embodiment 10. The method of Embodiment 9, wherein the target-hybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs:38, 39, 43, 44, and 53.

Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID N Os :80-82 and 85-100.

Embodiment 12. The method of any one of Embodiments 1 to 10, wherein the target-hybridizing sequence of (b) is contained in SEQ ID NO: 170 and comprises the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

Embodiment 13. The method of Embodiment 12, wherein the target-hybridizing sequence of (b) comprises the sequence of SEQ ID NO:171.

Embodiment 14. The method of Embodiment 13, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98.

Embodiment 15. The method of Embodiment 12, wherein the target-hybridizing sequence of (b) comprises the sequence of SEQ ID NO: 172.

Embodiment 16. The method of Embodiment 15, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs:80, 82, 85, and 87-100.

Embodiment 17. The method of any one of the preceding Embodiments, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b).

Embodiment 18. The method of Embodiment 17, wherein the promoter sequence is a T7 promoter sequence.

Embodiment 19. The method of Embodiment 18, wherein the T7 promoter sequence is SEQ ID NO:179.

Embodiment 20. The method of Embodiment 19, wherein the amplification oligomer of (b) comprises a sequence selected from the group consisting of SEQ ID NOs:57-59 and 62-77. X '

Embodiment 21. The method of Embodiment I, wherein the target-hybridizing sequences of (a) and (b), respectîvely, are (A) SEQ ID NO:30 and SEQ ID NO:82;

(B) SEQ ID NO:33 and SEQ ID NO:82;

(C) SEQ ID NO:49 and SEQ ID NO:82;

(D) SEQ ID NO:21 and SEQ ID NO:89 (E) SEQ ID NO:30 and SEQ IDNO:89;

(F) SEQ ID NO:33 and SEQ ID NO:89;

(G) SEQ ID NO:49 and SEQ IDNO:89;

(H) SEQ ID NO:21 and SEQ ID NO:92;

(I) SEQ ID NO:3Ü and SEQ ID NO:92;

(J) SEQ ID NO:21 and SEQ ID NO:94;

(K) SEQ ID NO:34 and SEQ ID NO:94;

(L) SEQ ID NO:53 and SEQ ID NO:94;

(M) SEQ ID NO:21 and SEQ ID NO:95;

(N) SEQ ID NO:34 and SEQ ID NO :95; or (O) SEQ ID NO:53 and SEQ ID NO:95.

Embodiment 22. The method of Embodiment 21, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b).

Embodiment 23.

a T7 promoter sequence.

The method of Embodiment 22, wherein the promoter sequence is

Embodiment 24. The method of Embodiment 23, wherein the T7 promoter sequence is SEQ ID NO: 179.

Embodiment 25. The method of Embodiment 1, wherein the at least two amplification oligomers comprise first and second amplification oligomers as in (a).

Embodiment 26. The method of Embodiment 25, wherein the at least two amplification oligomers comprise first and second amplification oligomers as in (a)(ii).

Embodiment 27. The method of Embodiment 26, wherein the first amplification oligomer as in (a)(ii) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 167; and the second amplification oligomer as in (a)(ii) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 168.

Embodiment 28. The method of Embodiment 27, wherein the first amplification oligomer comprises the target-hybridizing sequence of SEQ ID NO:34 and the second amplification oligomer comprises the target-hybridizing sequence of SEQ ID NO:53.,

Embodiment 29. The method of Embodiment 25, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii).

Embodiment 3Û. The method of Embodiment 29, wherein the amplification oligomer as in (a)(i) comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO; 164 and comprises the sequence of SEQ ID NO: 165; and the amplification oligomer as in (a)(ii) comprises the sequence of SEQ ID NO: 167.

Embodiment 31. The method of Embodiment 30, wherein the amplification oligomer as in (a)(i) comprises the target-hybridizing sequence of SEQ ID NO:21 and the amplification oligomer as in (a)(ii) comprises the target-hybridizing sequence of SEQ ID NO:34.

Embodiment 32. The method of any one of Embodiments 1 and 25 to 31, wherein the at least two amplification oligomers comprise first and second amplification oligomers of (b).

Embodiment 33. The method of Embodiment 32, wherein each of the first and second amplification oligomers of (b) comprises a target-hybridizing sequence that is contained în the sequence of SEQ ID NO: 170 and comprises the sequence of SEQ ID NO:171 or SEQ ID NO: 172.

Embodiment 34. The method of Embodiment 33, wherein the first amplification oligomer as in (b) comprises the target-hybridizing sequence of SEQ ID NO:94 and the second amplification oligomer as in (b) comprises the target-hybridizing sequence of SEQ ID NO:95.

Embodiment 35. The method of any one of Embodiments 32 to 34, wherein each of the amplification oligomers of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b).

Embodiment 36. The method of Embodiment 35, wherein the promoter sequence is a T7 promoter sequence.

Embodiment 37. The method of Embodiment 36, wherein the T7 promoter sequence isSEQ ID NO:179.

Embodiment 38. The method of any one of the preceding Embodiments, further comprising purifying the target nucleic acid from other components in the sample before step (UEmbodiment 39. The method of Embodiment 38, wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target hybrîdizing sequence covalently attached to a sequence or moiety that binds to an immobîlized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consistîng of SEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 40. The method of Embodiment 39, wherein the capture probe oligomer target-hybridizing sequence is selected from the group consistîng of SEQ ID NOs:l 115, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 41. The method of Embodiment 39, wherein the purifying step comprises contacting the sample with at least two capture probe oligomers.

Embodiment 42. The method of Embodiment 41, wherein the at least two capture probe oligomers comprise a first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 19; and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20.

Embodiment 43. The method of any one of the preceding Embodiments, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oiigomer comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of Plasmodium species in said sample.

Embodiment 44. The method of Embodiment 43, wherein the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consistîng of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 45. The method of Embodiment 44, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consistîng of SEQ ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 46. The method of Embodiment 44, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ü) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 47. The method of Embodiment 46, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 48. The method of Embodiment 47, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:148155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 49. The method of Embodiment 46, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises SEQ ID NO:175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 50. The method of Embodiment 49, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 51. The method of Embodiment 44, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises a sequence selected from the group consisting of SEQ ID NO:177 and SEQ ID NO:178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 52. The method of Embodiment 51, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:131, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 53. The method of any one of Embodiments 43 to 52, wherein the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide residue member of the détection probe oligomer nucléotide sequence.

Embodiment 54. The method of Embodiment 43 to 53, wherein the amplification oligomer target-hybridizing sequence of (a), the amplification oligomer target-hybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO: 151 or its complément, or a

DNA équivalent or DNA/RNA chimeric of SEQ ID NO:151 or its complément;

(B) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 157 or its complément;

(C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO:155 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 155 or its complément;

(D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO; 150 or its complément;

(E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 155 or its complément;

(F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO; 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(H) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 148 or its complément;

(I) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO: 158 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 158 or its complément;

(K) SEQ ID NO:49, SEQ ID NO;89, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO:15Û or its complément;

(L) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO; 148 or its complément;

(M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 152 or its complément;

(N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 148 or its complément;

(O) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 148 or its complément;

(Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO; 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément;

(U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(V) SEQ ID NO;53, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément;

(X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(Y) SEQIDNO:21, SEQ IDNO:95, and SEQ IDNO:152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(2) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(AB) SEQ ID NO:34, SEQ ID NO;95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO; 157 or its complément;

(AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO; 148 or its complément;

(AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément; or (AE) SEQ ID NO;53, SEQ ID NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément.

Embodiment 55. The method of any one of Embodiments 43 to 54, wherein the détection probe oligomer comprises a détectable label.

Embodiment 56. The method of Embodiment 55, wherein the détectable label is a chemiluminescent label or a fluorescent label.

Embodiment 57. The method of Embodiment 56, wherein the détectable label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer.

Embodiment 58. The method of one any of the preceding Embodiments, wherein the detecting step (3) occurs during the amplîfying step (2).

Embodiment 59. The method of Embodiment 56 or 58, wherein the détection probe comprises a fluorescent label and a quencher.

Embodiment 60. The method of Embodiment 59, wherein the détection probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan détection probe.

Embodiment 61. The method of any one of Embodiments 43 to 60, wherein the détection probe further comprises a non-target-hybridizîng sequence.

Embodiment 62. The method of Embodiment 61, wherein the détection probe is a molecular torch or a molecular beacon.

Embodiment 63. The method of Embodiment 43, wherein the at least one détection probe oligomer comprises at least two détection probe oligomers.

Embodiment 64. The method of Embodiment 44, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers, wherein (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (î) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (îi) comprises the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and (B) the second détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO; 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 65. The method of Embodiment 64, wherein the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and the second détection probe oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 66. The method of any one of the preceding Embodiments, wherein the amplification reaction at step (2) is an isothermal amplification reaction.

Embodiment 67. The method of Embodiment 66, wherein the amplification reaction îs a transcription-medîated amplification (TMA) reaction.

Embodiment 68. The method of any one of the preceding Embodiments, wherein the amplification reaction is a real-time amplification reaction.

Embodiment 69. The method of any one of the preceding Embodiments, wherein the sample is a clinical sample.

Embodiment 70. The method of any one of the preceding Embodiments, wherein the sample is a blood sample.

Embodiment 71. The method of any Embodiment 70, wherein the sample is a lysed blood ceil sample, optionally wherein the sample is a lysed red blood cell sample.

Embodiment 72. The method of Embodiment 70, wherein the sample is a red blood cell sample.

Embodiment 73. A combination of at least two oligomers for determining the presence or absence of Plasmodium species in a sample, said oligomer combination comprising at least two oligomers for amplifying a target région of Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:

(a) an amplification oligomer comprising a target-hybridizing sequence (i) that îs from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO:163; or (ii) that îs from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; and (b) an amplification oiigomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO; 169 and comprises the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173.

Embodiment 74. The oligomer combination of Embodiment 73, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(i).

Embodiment 75. The oligomer combination of Embodiment 74, wherein the targethybridizing sequence of (a)(i) is selected from the group consistîng of SEQ ID NOs:21, 23-25, 32, 33,35, 54, and 55.

Embodiment 76. The oligomer combination of Embodiment 74, wherein the targethybridizing sequence of (a)(0 is contained in the sequence of SEQ ID NO: 164 and comprises the sequence ofSEQ IDNO:165.

Embodiment 77. The oligomer combination of Embodiment 76, wherein the targethybridizing sequence of (a)(i) is selected from the group consisting of SEQ ID NOs:21, 23-25, 32, 33, and 35.

Embodiment 78. The oligomer combination of Embodiment 73, wherein the at least two amplification ohgomers comprise the amplification oligomer of (a)(ii).

Embodiment 79. The oligomer combination of Embodiment 78, wherein the targethybridizing sequence of (a)(îî) comprises the sequence of SEQ ID NO:167.

Embodiment 80. The oligomer combination of Embodiment 79, wherein the targethybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs:28-31, 34, 40, 41, and 49-51.

Embodiment 81. The oligomer combination of Embodiment 78, wherein the targethybridizing sequence of (a)(iî) comprises the sequence of SEQ ID NO: 168.

Embodiment 82. The oligomer combination of Embodiment 81, wherein the targethybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs:38, 39, 43, 44, and 53.

Embodiment 83. The oligomer combination of any one of Embodiments 73 to 82, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs:80-82 and 85-100.

Embodiment 84. The oligomer combination of any one of Embodiments 73 to 82, wherein the target-hybridizing sequence of (b) is contained in SEQ ID NO: 170 and comprises the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

Embodiment 85. The oligomer combination of Embodiment 84, wherein the targethybridizing sequence of (b) comprises the sequence of SEQ ID NO:171.

Embodiment 86. The oligomer combination of Embodiment 85, wherein the targethybridizing sequence of (b) is selected from the group consisting ofSEQ ID NOs:81, 82, 85, 8790, 94, and 96-98.

Embodiment 87. The oligomer combination of Embodiment 84, wherein the targethybridizing sequence of (b) comprises the sequence ofSEQ ID NO: 172.

Embodiment 88. The oligomer combination of Embodiment 87, wherein the targethybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs:80, 82, 85, and 87-100.

Embodiment 89. The oligomer combination of any one of the preceding

Embodîments, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b).

Embodiment 90. The oligomer combination of Embodiment 89, wherein the promoter sequence is a T7 promoter sequence.

Embodiment 91. The oligomer combination of Embodiment 90, wherein the T7 promoter sequence is SEQ ID NO: 179.

Embodiment 92. The oligomer combination of Embodiment 91, wherein the amplification oligomer of (b) comprises a sequence selected from the group consisting of SEQ ID NOs:57-59 and 62-77.

Embodiment 93. The oligomer combination of Embodiment 73, wherein the targethybridizing sequences of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82;

(B) SEQ ID NO:33 and SEQ ID NO:82;

(C) SEQ ID NO:49 and SEQ ID NO:82;

(D) SEQ ID NO:21 and SEQ ID NO:89 (E) SEQ ID NO:30 and SEQ ID NO:89;

(F) SEQ ID NO:33 and SEQ ID NO:89;

(G) SEQ ID NO:49 and SEQ ID NO:89;

(H) SEQ ID NO:21 and SEQ ID NO:92;

(I) SEQ ID NO;30 and SEQ ID NO:92;

(J) SEQ ID NO:21 and SEQ ID NO:94;

(K) SEQ ID NO:34 and SEQ ID NO:94;

(L) SEQ ID NO:53 and SEQ ID NO:94;

(M) SEQ IDNO:21 and SEQ IDNO:95;

(N) SEQ ID NO:34 and SEQ ID NO:95; or (O) SEQ ID NO:53 and SEQ ID NO:95.

Embodiment 94. The oligomer combination of Embodiment 93, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequenee located 5' to the target-hybridizing sequenee of (b).

Embodiment 95. The oligomer combination of Embodiment 94, wherein the promoter sequenee is a T7 promoter sequenee.

Embodiment 96. The oligomer combination of Embodiment 95, wherein the T7 promoter sequenee is SEQ ID NO: 179.

Embodiment 97. The oligomer combination of Embodiment 73, wherein the at least two amplification oligomers comprise first and second amplification oligomers as in (a).

Embodiment 98. The oligomer combination of Embodiment 97, wherein the at least two amplification oligomers comprise first and second amplification oligomers as in (a)(îî).

Embodiment 99. The oligomer combination of Embodiment 98, wherein the first amplification oligomer as in (a)(ü) comprises a target-hybridizing sequenee comprising the sequenee of SEQ ID NO: 167; and the second amplification oligomer as in (a)(ii) comprises a target-hybridizing sequenee comprising the sequenee of SEQ ID NO: 168.

Embodiment 100. The oligomer combination of Embodiment 99, wherein the first amplification oligomer comprises the target-hybridizing sequenee of SEQ ID NO:34 and the second amplification oligomer comprises the target-hybridizing sequenee of SEQ ID NO:53.

Embodiment 101. The oligomer combination of Embodiment 97, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii).

Embodiment 102. The oligomer combination of Embodiment 101, wherein the amplification oligomer as in (a)(i) comprises a target-hybridizing sequenee that is contained in the sequenee of SEQ ID NO: 164 and comprises the sequenee of SEQ ID NO: 165; and the amplification oligomer as in (a)(ii) comprises the sequenee of SEQ ID NO: 167.

Embodiment 103. The oligomer combination of Embodiment 102, wherein the amplification oligomer as in (a)(i) comprises the target-hybridizing sequenee of SEQ ID NO:21 and the amplification oligomer as in (a)(ii) comprises the target-hybridizing sequenee of SEQ ID NO:34.

Embodiment 104. The oligomer combination of any one of Embodiments 73 and 97 to 103, wherein the at least two amplification oligomers comprise first and second amplification oiigomers of (b).

Embodiment 105. The oligomer combination of Embodiment 104, wherein each of the first and second amplification oligomers of (b) comprises a target-hybridizing sequence that is contained in SEQ ID NO:170 and comprises the sequence of SEQ ID NO:171 or SEQ ID NO:172.

Embodiment 106. The oligomer combination of Embodiment 105, wherein the first amplification oligomer as in (b) comprises the target-hybridizing sequence of SEQ ID NO:94 and the second amplification oligomer as in (b) comprises the target-hybridizing sequence of SEQ ID NO:95.

Embodiment 107. The oligomer combination of Embodiment 93, wherein each of the amplification oligomers of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b).

Embodiment 108. The oligomer combination of Embodiment 107, wherein the promoter sequence is a T7 promoter sequence.

Embodiment 109. The oligomer combination of Embodiment 108, wherein the T7 promoter sequence is SEQ ID NO:179.

Embodiment 110. The oligomer combination of any one of the preceding Embodiments, further comprising at least one capture probe oligomer comprising a targethybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 111. The oligomer combination of Embodiment 110, wherein the capture probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 112. The oligomer combination of Embodiment 110, wherein the oligomer combination comprises at least two capture probe oligomers.

Embodiment 113. The oligomer combination of Embodiment 112, wherein the at least two capture probe oiigomers comprise a first capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO: 19, or a DNA équivalent or DNA/RNA chimeric thereof; and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20, or a DNA équivalent or DNA/RNA chimeric thereof

Embodiment 114. The oligomer combination of any one of the preceding Embodiments, further comprising at least one détection probe oligomer comprising a targethybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifîable by the at least two amplification oligomers.

Embodiment 115. The oligomer combination of Embodiment 114, wherein the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ (D NO:178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 116. The oligomer combination of Embodiment 115, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 117. The oligomer combination of Embodiment 115, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 174 and SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 118. The oligomer combination of Embodiment 117, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence of SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 119. The oligomer combination of Embodiment 118, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:148-155 and 159, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 120. The oligomer combination of Embodiment 117, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 121, The oligomer combination of Embodiment 120, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID N Os: 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 122. The oligomer combination of Embodiment 115, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises a sequence selected from the group consisting of SEQ ID NO; 177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 123. The oligomer combination of Embodiment 122, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:131, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 124. The oligomer combination of any one of Embodiments 114to 123, wherein the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide residue member of the détection probe oligomer nucléotide sequence.

Embodiment 125. The oligomer combination of Embodiment 114 to 124, wherein the amplification oligomer target-hybridizing sequence of (a), the amplification oligomer targethybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO:3Û, SEQ ID NO:82, and SEQ ID NO:151 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO:151 or its complément;

(B) SEQ ID NO;30, SEQ ID NO:82, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 157 or its complément;

(C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 155 or its complément;

(D) SEQ ID NO:49, SEQ ID NO;82, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 150 or its complément;

(E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 155 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 155 or its complément;

(F) SEQ ID NO:21, SEQ ID NO;89, and SEQ ID NO; 148 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 148 or its complément;

(G) SEQ ID NO:21, SEQ ID NO:89, and SEQ TD NO; 152 or its complément, or a DNA équivalent or DNA/RNA chîmeric of SEQ ID NO: 152 or its complément;

(H) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(1) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO:I58 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO;158 or its complément;

(K) SEQ ID NO:49, SEQ ID NO:89, and SEQ ID NO: 150 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 150 or its complément;

(L) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 152 or its complément, or a

DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(O) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a

DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément;

(U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(V) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complément;

(X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(Y) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complément;

(AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complément;

(AB) SEQ ID NO:34, SEQ ÎD NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 157 or its complément;

(AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 148 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 148 or its complément;

(AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 152 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 152 or its complément; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 157 or its complément.

Embodiment 126. The oligomer combination of any one of Embodiments 114 to 125, wherein the détection probe oligomer comprises a détectable label.

Embodiment 127. The oligomer combination of Embodiment 126, wherein the détectable label is a chemiluminescent label or a fluorescent label.

Embodiment 128. The oligomer combination of Embodiment 127, wherein the détectable label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer.

Embodiment 129. The oligomer combination of Embodiment 127, wherein the détection probe comprises a fluorescent label and a quencher.

Embodiment 130. The oligomer combination of Embodiment 129, wherein the détection probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan détection probe.

Embodiment 131. The oligomer combination of any one of Embodiments 114to 130, wherein the détection probe further comprises a non-target-hybridizing sequence.

Embodiment 132. The oligomer combination of Embodiment 131, wherein the détection probe is a molecular torch or a molecular beacon.

Embodiment 133. The oligomer combination of Embodiment 114, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers.

Embodiment 134. The oligomer combination of Embodiment 115, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers, wherein (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (il) comprises the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and (B) the second détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 135. The oligomer combination of Embodiment 134, wherein the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and the second détection probe oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 136. A détection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, said détection probe oligomer comprising a target-hybridizing sequence that is from about 13 to about 40 nucléotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target région amplifîable by an oligomer combination comprising first and second Plasmodiumspecîfic amplification oligomers, wherein (a) the first amplification oligomer comprises a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO:I62, and comprises the sequence of SEQ ID NO: 163, or (ii) that is from about 14 to about 25 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; and (b) the second amplification oligomer comprises a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173.

Embodiment 137. The détection probe oligomer of Embodiment 136, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO:!96 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 138. The détection probe oligomer of Embodiment 137, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimencs thereof.

Embodiment 139. The détection probe oligomer of Embodiment 137, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA chimencs thereof.

Embodiment 140. The détection probe oligomer of Embodiment 139, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence of SEQ TD NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 141. The détection probe oligomer of Embodiment 140, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:148-155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 142. The détection probe oligomer of Embodiment 139, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 143. The détection probe oligomer of Embodiment 142, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 144. The détection probe oligomer of Embodiment 137, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 145, The détection probe oligomer of Embodiment 144, wherein the détection probe oligomer target-hybridizing sequenee is selected from the group consisting of SEQ ID NOs:l31, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof,

Embodiment 146. The détection probe oligomer of any one of Embodiments 136 to 145, wherein the détection probe oligomer comprises a 2' methoxy modification on at least one of a nucléotide residue member of the détection probe oligomer nucléotide sequenee.

Embodiment 147. The détection probe oligomer of any one of Embodiments 136 to 146, wherein the détection probe oligomer comprises a label,

Embodiment 148. The détection probe oligomer of Embodiment 147, wherein the label is a chemiluminescent label or a fluorescent label.

Embodiment 149. The détection probe oligomer of Embodiment 148, wherein the label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the détection probe oligomer.

Embodiment 150. The détection probe oligomer of Embodiment 148, wherein the détection probe comprises a fluorescent label and a quencher.

Embodiment 152. The détection probe oligomer of Embodiment 150, wherein the détection probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan détection probe.

Embodiment 153. The détection probe oligomer of any one of Embodiments 136 to 152, wherein the détection probe further comprises a non-target-hybridizing sequenee.

Embodiment 154. The détection probe oligomer of Embodiment 153, wherein the détection probe is a molecular torch or a molecular beacon.

Embodiment 155. A combination of at least two oligomers for detecting a Plasmodium species target nucleic acid in a sample, said oligomer combination comprising at least two détection probe oligomers of Embodiment 136.

Embodiment 156. The oligomer combination of Embodiment 155, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers, wherein (A) the first détection probe oligomer comprises a target-hybridizing sequenee that (i) is contained in the sequenee of SEQ ID NO: 197 or its complément, or a DNA équivalent or

DNA/RNA chimeric thereof, and (ii) comprises the sequence ofSEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and (B) the second détection probe oligomer comprises a target-hybridizing sequence that (i) is contained in the sequence ofSEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 157. The oligomer combination of Embodiment 156, wherein the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and the second détection probe oligomer comprises a target-hybridizing sequence selected from the group consisting ofSEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 158. A capture probe oligomer for specifically îsolating Plasmodium species nucleic acid from a sample, said capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 159. The capture probe oligomer of Embodiment 158, wherein the capture probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

Embodiment 160. A combination of at least two oligomers for specifically îsolating Plasmodium species nucleic acid from a sample, wherein the oligomer combination comprises at least two capture probe oligomers of Embodiment 158.

Embodiment 161. The oligomer combination of Embodiment 160, wherein the at least two capture probe oligomers comprise a first capture probe oligomer comprising the target-hybridizing sequence ofSEQ ID NO: 19, or a DNA équivalent or DNA/RNA chimeric thereof; and a second capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20, or a DNA équivalent or DNA/RNA chimeric thereof.

Embodiment 162. A kit comprising the combination of at least two oligomers according to any one of Embodiments 73 to 135, 155 to 157, 160, and 161.

Embodiment 163. A reaction mixture comprising the combination of at least two oligomers according to any one of Embodiments 73 to 135, 155 to 157, 160, and 161.

Embodiment 164. Use of the combination of at least two oligomers according to any one of Embodiments 73 to 135 for specifically amplifying Plasmodium species nucleic acid in a sample.

Embodiment 165. Use of the détection probe oligomer or oligomer combination according to any one of Embodiments 136 to 157 for specifically detecting Plasmodium species nucleic acid in a sample.

Embodiment 166. Use of the capture probe oligomer or oligomer combination according to any one of Embodiments 158 to 161 for specifically capturing Plasmodium species nucleic acid from a sample.

Embodiment 167. A method for specifically detecting Plasmodium species nucleic acid in a sample, said method comprising:

( 1 ) contacting a sample, said sample suspected of containing Plasmodium species nucleic acid, with at least two oligomers for amplifying a target région of a Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:

(a) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b) an amplification oligomer comprising a target-hybridizing sequence that is contained in SEQ ID NO: 188 and comprises the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO: 182;

(2) performing an in vitro nucleic acid amplification reaction, wherein any Plasmodium target nucleic acid présent in said sample is used as a template for generating an amplification product; and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of Plasmodium species target nucleic acid in said sample.

Embodiment 168. The method of Embodiment 167, wherein the target-hybridizing sequence of (a) is selected from the group consisting of SEQ ID NOs:37, 46, 183, and 184.

Embodiment 169. The method of Embodiment 168, wherein the target-hybridizing sequence of (a) is contained in the sequence of SEQ ID NO: 186.

Embodiment 170. The method of Embodiment 169, wherein the target-hybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184.

Embodiment 171, The method of any one of Embodiments 167 to 170, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs:83, 84, and 182.

Embodiment 172. The method of Embodiment 167, wherein the target-hybridizing 5 sequences of (a) is SEQ ID NO:183 or SEQ ID NO:184 and the target-hybridizing sequence of (b) is SEQ ID NO:182.

Embodiment 173. The method of any one of Embodiments 167 to 172, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer comprisîng a target-hybridizing sequence configured to 10 specifîcally hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of Plasmodium species in said sample.

Embodiment 174. The method of Embodiment 173, wherein the détection probe oligomer target-hybridizing sequence is at least about 13 nucléotides in length and is (i) 1S contained in the sequence of SEQ ID NO: 189 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO:190 and SEQ ID NO:191, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 175. The method of Embodiment 174, wherein the détection probe 20 oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 125130 and 143, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 176. The method of Embodiment 173, wherein the amplification oligomer target-hybridizing sequence of (a), the amplification oligomer target-hybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are 25 (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a

DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément;

(B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 127 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 127 or its complément;

(C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or its complément, or a 30 DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 128 or its complément;

(D) SEQ ID NO:183, SEQ ID NO;182, and SEQ ID NO: 143 or its complément, or a DNA équivalent or DNA/RNA chimeric ofSEQ ID NO: 143 or its complément;

(E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 129 or its complément; or (F) SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a

DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément.

Embodiment 177. A combination of at least two oligomers for determining the presence or absence of Plasmodium species in a sample, said oligomer combination comprising at least two oligomers for amplifying a target région of Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:

(a) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b) an amplification oligomer comprising a target-hybridizing sequence that is contained in SEQ ID NO:188 and comprises the sequence of SEQ ID NO:83, 15 SEQ ID NO:84, or SEQ ID NO: 182.

Embodiment 178. The oligomer combination of Embodiment 177, wherein the targethybridizing sequence of (a) is selected from the group consisting of SEQ ID NOs:37, 46, 183, and 184.

Embodiment 179. The oligomer combination of Embodiment 178, wherein the target20 hybridizing sequence of (a) is contained in the sequence of SEQ ID NO: 186.

Embodiment 180. The oligomer combination of Embodiment 179, wherein the targethybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184.

Embodiment 181. The oligomer combination of any one of Embodiments 177 to 180, wherein the target-hybridizing sequence of (b) is selected from the group consisting of SEQ ID 25 NOs:83, 84, and 182.

Embodiment 182. The oligomer combination of Embodiment 177, wherein the targethybridizing sequences of (a) is SEQ ID NO: 183 or SEQ ID NO: 184 and the target-hybridizing sequence of (b) is SEQ ID NO: 182.

Embodiment 183. The oligomer combination of any one of Embodiments 177 to 182, 30 further comprising at least one détection probe oligomer comprising a target-hybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifîable by the at least two amplification oligomers.

100

Embodiment 184. The oligomer combination of Embodiment 183, wherein the détection probe oligomer target-hybridizing sequence is at least about 13 nucléotides în length and îs (i) contained in the sequence of SEQ ID NO;189 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 190 and SEQ ID NO: 191, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

Embodiment 185. The oligomer combination of Embodiment 174, wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 125-130 and 143, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

Embodiment 186. The oligomer combination of Embodiment 173, wherein the amplification oligomer target-hybridizing sequence of (a), the amplification oligomer targethybridizing sequence of (b), and the détection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément;

(B) SEQ ID NO: 183, SEQ IDNO:182, and SEQ ID NO: 127 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 127 or its complément;

(C) SEQ ID NO: 183, SEQ IDNO:182, and SEQ ID NO: 128 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 128 or its complément;

(D) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO; 143 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 143 or its complément;

(E) SEQ ID NO; 183, SEQ ID NO: 182, and SEQ ID NO: 129 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 129 or its complément; or (F) SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complément, or a DNA équivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complément.

Embodiment 187. A détection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, said détection probe oligomer comprising a target-hybridizing sequence that is at least about 13 nucléotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target région amplifîable by an oligomer combination comprising first and second Plasmodium-specïÎic amplification oiigomers, wherein

101 (a) the first amplification oligomer comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b) the second amplification oligomer comprises a target-hybridizing sequence that is contained in SEQ ID NO: 188 and comprises the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO: 182.

Embodiment 188, A kit comprising the combination of at least two oligomers according to any one of Embodîments 177 to 186.

Embodiment 189. A reaction mixture comprising the combination of at least two oligomers according to any one of Embodîments 177 to 186.

Embodiment 190, Use of the combination of at least two oligomers according to any one of Embodîments 177 to 186 for specifically amplifÿing Plasmodium species nucleic acid in a sample.

Embodiment 191. Use of the détection probe oligomer or oligomer combination according to any one of Embodîments 177 to 187 for specifically detecting Plasmodium species nucleic acid in a sample.

From the foregoing, it will be appreciated that, although spécifie embodîments of the invention hâve been described herein for purposes of illustration, varions modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended daims. AU publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for ail purposes.

Claims (2)

1. । · A method for specifically detecting Plasmodium species nucleic acid in a sample, said method comprising:

   ( I ) contacting a sample, said sample suspected of containing Plasmodium species nucleic acid, w,th at least two oligomers for amplilying _a target _{region of a Plasmo<Ulm species} nucleic acid, wherein the al to two amplification oligomers comprise:

   (a) an amplification oligomer comprising a target-hybridizing sequence (i) that is from aboul 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163; or (ii) thaï is from abou. 14 to abou! 25 conliguous nucléotides in length, is contained in the sequence ofSEQ ID NO: 166, and comprises the sequence ofSEQ ID NO:167 or SEQ ID NO:168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ IDNO:169and comprises the sequence ofSEQ IDNO;17î,SEQ ID NO: 172, or SEQ ID NO; 173;
2. (2) performing an i„ _vilro nucleic acid amplification reaction, wherein any Plasmodium target nucleic acid présent in said sample is used as a tempiate for generaüng an amplification product; and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of Plasmodium species target nucleic acid in said sample.

   2. The method of claim !, wherein the at to two amplification oligomers comprise the amplification oligomer of (a)(i).

   3. The method of claim 2, wherein the target-hybridizing sequence of (a)(i) is selected from the group consisting ofSEQ )D NOs:21, 23-25, 32, 33, 35, 54, and 55, or wherein the target-hybridizing sequenee of (a)(i) is contained in the sequenee of SEQ ID NO: 164, comprises the sequenee ofSEQ ID NO: 165, and is optionally selected from the group consisting ofSEQ ID NOs:2l, 23-25, 32, 33, and 35,

   4. The method oi claîm I, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(ii).

   5, 1 he method of claîm 4, wherein the target-hybridizing sequenee of (a)(ii) comprises the sequenee ol SEQ ID NO: 167 and is optionally selected from the group consisting of SEQ JD NOs:28-31,34, 40, 41, and 49-51.

   6. The method of claîm 4, wherein the target-hybridizing sequenee of (a)(ii) comprises the sequenee ofSEQ ID NO: 168 and is optionally selected from the group consisting ofSEQ ID NOs:38, 39, 43, 44, and 53.

   7· The method of any one of daims 1 to 6, wherein the target-hybridizing sequenee of (b) is selected from the group consisting ofSEQ ID NOs:80-82 and 85-100, or wherein the target-hybridizing sequenee of (b) is contained in SEQ ID NO:170, comprises the sequenee ofSEQ ID NO:171 or SEQ ID NO: 172, and is optionally selected from (i) the group consisting ofSEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98 or (ii) the group consisting ofSEQ ID NOs:80, 82, 85, and 87-100.

   8. The method of any one of the preccding daims, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to die target-hybridizing sequenee of (b), optionally wherein the promoter sequenee is a T7 promoter sequenee, optionally wherein the amplification oligomer of (b) comprises a sequenee selected from the group consisting of SEQ ID NOs:57-59 and 62-77.

   9. The method of claîm I, wherein the target-hybridizing sequences of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82;

   ie4 (B) SEQ ID NO:33 and SEQ JD NO:82;

   (C) SEQ ÏD NO:49 and SEQ ID NO:82;

   (D) SEQ ID NO:21 and SEQ ID NO:S9 (E) SEQ ID NO:30 and SEQ ID NO:89;

   5 (F) SEQ ID NO:33 and SEQ ID NO:89;

   (G) SEQ ID NO:49 and SEQ ID NO:89;

   (H) SEQ ID NO:21 and SEQ ID NO:92;

   (I) SEQ ID NO:30 and SEQ )D NO:92;

   (J) SEQ ID NO:21 and SEQ ID NO:94;

   10 (K) SEQ ID NO:34 and SEQ ID NO:94;

   (L)' SEQ ID NO:53 and SEQ ID NO’:94;

   (M) SEQ ID NO:21 and SEQ ID NO:95;

   (N¹) SEQ ID NO:34 and SEQ ID NO:95; or (O) SEQ ID NO:53 and SEQ ID NO:95.

   10. The method of claim 1, wherein the at least two amplification oligomers comprise first and second amplification oligomers as in (a)(ii), optionally wherein the first amplification oligomer as in (a)(H) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 167, optionally wherein the first amplification oligomer 20 target-hybridizing sequence is SEQ ID NO:34; and the second amplification oligomer as in (a)(ii) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 168, optionally wherein the second amplification oligomer target-hybridizing sequence is SEQ ID NO:53.

   ^{25 ] 1} ’ ^{The meÎ}hod of claim 1, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (aXii), optionally wherein the amplification oligomer as in (a)(i) comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 164 and comprises the sequence of SEQ ID NO: 165,

   30 optionally wherein the amplification oligomer as in (a)(i) comprises the target-hybridizing i sequence of SEQ IDJ4O:21; and the amplification oligomer as in (a)(îi) comprises the sequence ofSEQ ID NO: 167, optionally wherein the amplification oligomer as in (a)(ii) comprises the target-hybridizing sequence of SEQ ID NO:34.

   12. The method ofany one of daims 1,10, and 1 ], wherein the at least two amplification olîgomers comprise first and second amplification oligomers of (b), optionally wherein each of the first and second amplification oligomers of (b) comprises a targethybridizing sequence that is contained in the sequence ofSEQ ID NO: 170 and comprises the sequence ofSEQ ID NO: 171 or SEQ ID NO: 172, optionally wherein the tlrst amplification oligomer as in (b) comprises the targethybridizing sequence ofSEQ ID NO:94 and the second amplification oligomer as in (b) comprises the target-hybridizing sequence ofSEQ ID NO:95.

   13. Ί he method of any one of the preceding claims, further comprising purifying the target nucleic acid from other components in the sample before step (1), optionally wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consisting ofSEQ ID NOs:l 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof

   14. The method of any one of the preceding claims, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one détection probe oligomer comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is detennincd, thereby indicating the presence or absence of Plasmodium species in said sample.

   15. The method of claim 14, wherein the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence ofSEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consistîng of SEQ ID NO: 175, SEQ ID NO: 176, SEQ IDNO:I77, and SEQ 1DNO:178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof, optionally wherein the détection probe oiigomer target-hybridizing sequence is selected 5 from the group consistîng of SEQ IDNOs: 131, 132, 135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

   16. The method of claim 1 5, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA

   10 équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA chimerics thereof, optionally wherein (a) the détection probe oiigomer target-hybridizing sequence (ii) comprises SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally 15 wherein the détection probe oligomer target-hybridizing sequence is selected from the group consistîng of SEQ ID NOs: 148-155 and 159, including compléments, DNA équivalents, and DNA/RNA chimerics thereof, or (b) the détection probe oiigomer target-hybridizing sequence (ii) comprises SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally 20 wherein the détection probe oiigomer target-hybridizing sequence îs selected from the group consistîng of SEQ ID NOs:147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

   17. The method of claim 15, wherein the détection probe oligomer target-hybridizing 25 sequence (ii) comprises a sequence selected from the group consistîng of SEQ ID NO:177 and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA chimerics thereof, optionally wherein the détection probe oligomer target-hybridizing sequence is selected from the group consistîng of SEQ IDNOs:131, 132, 135, 140, 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

   1^7

   18. The method of claim 15, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers, wherein (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and (B) the second détection probe oligomer comprises a target-hybridizing sequence that is (î) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO; 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the second détection probe oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ JD NO: 152, including compléments, DNA équivalents, and DNA/RNA chimerics thereof.

   19. The method of any one of the preceding daims, wherein the amplification reaction at step (2) is an isothermal amplification reaction, optionally wherein the amplification reaction is a transcription-medîated amplification (TMA) reaction, and optionally wherein the amplification reaction is a real-time amplification reaction.

   20. A combination of at least two oiigomers for determining the presence or absence of Plasmodium species in a sample, said oligomer combination comprising at least two oligomers for amplifying a target région of Plasmodium species target nucleic acid, wherein the at least two amplification oiigomers comprise:

   (a) an amplification oligomer comprising a target-hybridizing sequence (i) that is from about 14 to about 20 conliguous nucléotides in length. is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163; or

   1ê8 (ii) that is from about 14 to about 25 contiguous nucléotides in length, îs contained in the sequence ofSEQ ID NO: 166, and comprises the sequence ofSEQ ID NO: 167 or SEQ ID NO; 168; and (b) an amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and comprises the sequence ofSEQ ID NO:171, SEQ ID NO: 172, or SEQ IDNO:173.

   21. The oligomer combination of claim 20, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(i).

   22. The oligomer combination ofclaim 21, wherein the target-hybridizing sequence of(a)(i) is selected from the group consisting ofSEQ ID NOs:21,23-25, 32, 33, 35, 54, and 55, or wherein the target-hybridizing sequence of (a)(i) is contained in the sequence ofSEQ ID NO: 164, comprises the sequence ofSEQ ID NO: 165, and is optionally selected from the group consisting ofSEQ IDNOs:21, 23-25, 32, 33, and 35.

   23. The oligomer combination of claim 20, wherein the at least two amplification oligomers comprise the amplification oligomer of (a)(ii).

   24. The oligomer combination ofclaim 23, wherein the target-hybridizing sequence of (a)(ü) comprises the sequence of SEQ ID NO:167 and is optionally selected from the group consisting of SEQ ID NOs:28-31,34, 40, 41, and 49-51.

   25. I he oligomer combination of claim 23, wherein the target-hybridizing sequence of (a)(iî) comprises the sequence ofSEQ ID NO: 168 and is optionally selected from the group consisting of SEQ ID NOs:38, 39, 43, 44, and 53.

   26. The oligomer combination of any one of daims 20 to 25, wherein the targethybridizing sequence of (b) is selected from the group consisting ofSEQ ID NOs:80-82 and 85100,

   1S9 or wherein the target-hybridizing sequence of (b) is contained in SEQ ID NO: 170, comprises the sequence of SEQ ID NO: 171 or SEQ ID NO:172, and is optionally seiected from (i) the group consisting of SEQ ID NOs:81,82, 85, 87-90, 94, and 96-98 or (ii) the group consisting of SEQ ID NOs:80, 82, 85, and 87-100.

   27. The oligomer combination of any one of the preceding claims, wherein the amplification oligomer of (b) is a promoter primer or promoter provider further comprising a promoter sequence located 5' to the target-hybridizing sequence of (b), optionally wherein the promoter sequence is a T7 promoter sequence, optionally wherein the amplification oligomer of (b) comprises a sequence seiected from the group consisting of SEQ ID NOs:57-59 and 62-77.

   28. The oligomer combination of claim 20, wherein the target-hybridizins sequences of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82;

   (B) SEQ ID NO:33 and SEQ ID NQ:82;

   (C) SEQ ID NO:49 and SEQ ID NO:82;

   (D) SEQ ID NO:21 and SEQ ID NO:89 (E) SEQ ID NO:30 and SEQ ID NO:89;

   (F) SEQ ID NO:33 and SEQ ID NO:89;

   (G) SEQ ID NO:49 and SEQ ID NO:89;

   (H) SEQ ID NO:21 and SEQ ID NO:92;

   (I) SEQ ID NO:30 and SEQ ID NO:92;

   (J) SEQ ID NO:21 and SEQ ID NO:94;

   (K) SEQ ID NO:34 and SEQ ID NO;94;

   (L) SEQ ID NO:53 and SEQ ID NO:94;

   (M) SEQ ID NO:21 and SEQ ID NO:95;

   (N) SEQ ID NO:34 and SEQ ID NO:95; or (O) SEQ ÏDNO:53 and SEQ IDNO:95.

   29. The oligomer combination of claim 20, wherein the at least two amplification oligomers comprise First and second amplification oligomers as in (a)(ii), optionally wherein il® the first amplification oligomer as in (a)(îi) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 167, optionally wherein the first amplification oligomer target-hybridizing sequence is SEQ ID NO:34; and the second amplification oligomer as in (a)(ii) comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 168, optionally wherein the second amplification oligomer target-hybridizing sequence is SEQ ID NO:53.

   30. The oligomer combination of claim 20, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii), optionally wherein the amplification oligomer as in (a)(i) comprises a target-hybridizing sequence that is contained in the sequence ofSEQ ID NO: 164 and comprises the sequence ofSEQ 1DNO:I65, optionally wherein the amplification oligomer as in (a)(i) comprises the target-hybridizing sequence ofSEQ ID NO:21; and the amplification oligomer as in (a)(ii) comprises the sequence of SEQ ID NO: 167, optionally wherein the amplification oligomer as in (a)(ii) comprises the target-hybridizing sequence ofSEQ ID NO:34.

   31. The oligomer combination of any one of daims 20, 29, and 30, wherein the at least two amplification oligomers comprise first and second amplification oligomers of (b), optionally wherein each of the first and second amplification oligomers ot (b) comprises a targethybridizing sequence that is contained in SEQ ID NO: 170 and comprises the sequence ofSEQ IDNO:171 or SEQ ID NO: 172, optionally wherein the first amplification oligomer as in (b) comprises the targethybridizing sequence ofSEQ ID NO:94 and the second amplification oligomer as in (b) comprises the target-hybridizing sequence of SEQ ID NO:95.

   32. The oligomer combination of any one of the preceding daims, further comprising at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected

   111 from the group consisting of SEQ JD NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

   33. The oligomer combination of any one of the preceding cl ai ms, further comprisîng at least one détection probe oligomer comprisîng a target-hybridizing sequence configured to specifîcally hybridize to a Plasmodium species amplicon amplifîable by the at least two amplification oligomers.

   34. The oligomer combination of claim 33, wherein the détection probe oligomer target-hybridizing sequence is from about 13 to about 40 nucléotides in length and is (i) contained in the sequence ofSEQ JD NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ IDNO:175, SEQ IDNO:J76, SEQ ID NO: 177, and SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof, optionally wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting ofSEQ IDNOs:131, 132,135, 140, 145, 147-157, and 159-161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

   j5. Ί he oligomei combination of claim 34, wherein the détection probe oligomer target-hybridizing sequence is (i) contained in the sequence ofSEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 174 and SEQ ID NO: 175, including compléments, DNA équivalents, and DNA/RNA équivalents thereof, optionally wherein (a) the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence of SEQ ID NO: 174 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting ofSEQ JD NOs: 148-155 and 159, including compléments, DNA équivalents, and DNA/RNA équivalents thereof, or (b) the détection probe oligomer target-hybridizing sequence (ii) comprises the sequence ofSEQ ID NO: 175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the détection probe oligomer target-hybridizing sequence is selected

   112 from the group consisting of SEQ IDNOs: 147, 156, 157, 160, and 161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof

   36. The oligomer combination of claîm 34, wherein the détection probe oligomer target-hybridizing sequence (ii) comprises a sequence selected from the group consisting of SEQ ID NO:177 and SEQ JD NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof, optionally wherein the détection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157,160, and 161, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

   37. The oligomer combination of claîm 34, wherein the at least two détection probe oligomers comprise first and second détection probe oligomers, wherein (A) the first détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: ] 97 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO:175 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the first détection probe oligomer comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof; and (B) the second détection probe oligomer comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO: 197 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, and (ii) comprises the sequence of SEQ ID NO: 176 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof, optionally wherein the second détection probe oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

   38. A détection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, said détection probe oligomer comprising a target-hybridizing sequence that is from about 13 to about 40 nucléotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target région amplifîable by an

   113 oligomer combination comprising first and second Plasmodium-specifïc amplification oligomers, wherein (a) the first amplification oligomer comprises a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous nucléotides in length, is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163, or (ii) that is from about 14 to about 25 contiguous nucléotides în length, is contained in the sequence of SEQ 1DNO:166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO : 168; and (b) the second amplification oligomer comprises a target-hybridizing sequence that is from about 15 to about 33 contiguous nucléotides in length, is contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO: 171, SEQ IDNO:172, or SEQ IDNO:173;

   wherein the détection probe oligomer target-hybridizing sequence is (i) contained în the sequence of SEQ ID NO: 196 or its complément, or a DNA équivalent or DNA/RNA chimeric thereof. and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO: 178, including compléments, DNA équivalents, and DNA/RNA équivalents thereof.

   39. A combination of at least two oligomers for detecting a Plasmodium species target nucleic acid in a sample, saîd oligomer combination comprising at least two détection probe oligomers of daim 38.

   40. A method for isolating a Plasmodium species nucleic acid from a sample, said method comprising contacting the sample with at least one capture probe oligomer, wherein the at least one capture probe oligomer comprises a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof, and separating the Plasmodium species nucleic acid from other components in the sample using the immobilized probe.

   d k

   41. A combination of at least two oiigomers for specifically isolatin g Plasmodium species nucleic acid from a sample, wherein the oligomer combination comprises at least two capture probe oiigomers wherein each capture probe oligomer comprises a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucléotides in length and comprises a sequence selected from the group consisting of SEQ IDNOs:] 1-15, 17, 19, and 20, including DNA équivalents and DNA/RNA chimerics thereof.

   42. A kit comprising the combination of at least two oiigomers according to any one of daims 20 to 37, 39, and 41.

   43. A reaction mixture comprising the combination of at least two oiigomers according to any one of daims 20 to 37, 39, and 41.

   44. A method for specifically detecting Plasmodium species nucleic acid in a sample, said method comprising:

   (1) contact ing a sample, said sample suspected of containing Plasmodium species nucleic acid, with at least two oiigomers for amplifying a target région of a Plasmodium species target nucleic add, wherein the at least two amplification oligomers comprise:

   (a) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187; and (b) an amplification oligomer comprising a target-hybridizing sequence that is contained in SEQ ID NO: 188 and comprises the sequence ofSEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182;

   (2) performing an in vitro nucleic acid amplification reaction, wherein any Plasmodium target nucleic acid présent in said sample is used as a template for generating an amplification product; and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of Plasmodium species target nucleic acid in said sample.

   115

   45. A combination of at least two oligomers for determining the presence or absence of Plasmodium species in a sample, said oligomer combination comprising at least two oligomers for amplifying a target région of Plasmodium species target nucleic acid, wherein the at least two amplification oligorners comprise:

   5 (a) an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID N0:187; and (b) an amplification oligomer comprising a target-hybridizing sequence that is contained in SEQ ID NO: 188 and comprises the sequence ofSEQ ID NO:83, ¹⁰ SEQ ID NO:84, or SEQ IDNO:182.

   46. A détection piobe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, said détection probe oligomer comprising a target-hybridizing sequence that is at least about ] 3 nucléotides in length and configured to specifically hybridize to a target

   15 sequence contained within a Plasmodium species target région amplifîable by an oligomer combination comprising first and second Plasmodium-spécifie amplification oligomers, wherein (a) the first amplification oligomer comprises a target-hybridizing sequence that is contained in the sequence ofSEQ ID NO: 185 and comprises the sequence ofSEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and ²⁰ (^b) ^Îhe second amplification oligomer comprises a target-hybridizing sequence that is contained in SEQ ID NO: 188 and comprises the sequence ofSEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO: 182.

   47. A kit comprising the combination of at least two oligomers according to claim 45. >5

   48. A reaction mixture comprising the combination of at least two oligomers according to claim 45.