WO2000043545A2

WO2000043545A2 - Detection of drug resistant organisms

Info

Publication number: WO2000043545A2
Application number: PCT/US1999/029517
Authority: WO
Inventors: Yen Ping Liu; Nurith Kurn
Original assignee: Dade Behring Inc.
Priority date: 1999-01-19
Filing date: 1999-12-14
Publication date: 2000-07-27
Also published as: WO2000043545A3

Abstract

A method is disclosed for detecting drug resistance in a strain of an organism. In the method the presence of at least one mutation in a predetermined region within the gene of the strain is detected. The predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the organism. To detect the mutation, a complex is formed comprising the predetermined region of the gene of the organism and the corresponding region of the gene of the wild type organism in double stranded form. Each member of at least one pair of non-complementary strands within the complex has a label. The association of the labels within the complex is detected wherein the association of the labels in the complex is related to the presence of the mutation. The presence of the mutation is related to the drug resistance of the strain.

Description

DETECTION OF DRUG RESISTANT ORGANISMS CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of U.S. Patent Application Serial No. 09/233,996 filed January 19, 1999, the disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside tnphosphates of high specific activity and the 32p labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest.

Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.

One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.

When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.

A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the hybridization sites on the DNA sequence complementary to the 5' ends of the oligonucleotide primers.

Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q- beta-replicase method. Regardless of the amplification used, the amplified product must be detected. Genetic recombination involves the exchange of DNA strands between two related

DNA duplexes. The branch point between two duplex DNA's that have exchanged a pair of strands is thought to be an important intermediate in homologous recombination. This branch point is otherwise referred to as the Holliday junction. Movement of the Holliday junction by branch migration can increase or decrease the amount of genetic information exchanged between homologues. In vivo strand exchange is protein mediated, unlike the spontaneous migration that occurs in vitro.

Certain mycobacteria are causative agents of disease. Cases of mycobacterial infections are increasing in the United States. Of particular concern is tuberculosis, the etiological agent of which is M. tuberculosis. Many of these new cases are related to the AIDS epidemic, which provides an immune compromised population that is particularly susceptible to infection by Mycobacteria. Other mycobacterial infections are also increasing as a result of the increase in available immune compromised patients. Mycobacte um avium, Mycobacterium kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in HIV infected and other immune compromised patients.

Multidrug resistance is one factor in the tuberculosis problem. An increase in the frequency of Mycobacterium tuberculosis strains resistant to one or more anti- mycobacterial agents has been reported. Immunocompromised patients such as those infected with human immunodeficiency virus (HIV-1), who are not infected with M. tuberculosis, are frequently infected with other mycobacterial strains, which are often resistant to the drugs used to treat M. tuberculosis. Consequently, it is important to accurately determine drug sensitivities and identification of the mycobacteria species. Additionally, many new cases of M. tuberculosis are resistant to one or more of the primary anti-tuberculosis drugs, namely, isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide. Thus, the determination of drug resistance has become central concern during the diagnosis of mycobacterial diseases.

Methods used to determine drug sensitivity information include culture methods. Mycobacteria are judged to be resistant to particular drugs by use of either the standard proportional plate method or minimal inhibitory concentration method. More recently, approaches to determine drug sensitivity based on molecular genetics have been developed.

Current antibiotic susceptibility test results suggest that rifampin resistance can be used as a predictive marker of multidrug resistance in M. tuberculosis. In general, resistance to rifampin correlates well with resistance to three or more other anti-tuberculosis drugs. Culture-based antibiotic susceptibility testing of M. tuberculosis is tedious and lengthy, due to slow growth rate. The recent development of molecular techniques for the genotypic detection of mycobacterial infection and determination of antibiotic susceptibility are of utmost clinical importance.

Most drug resistance phenotypes in M. tuberculosis appear to arise as a consequence of chromosomal mutations. Multidrug resistance appears to be due to stepwise accumulation of mutations conferring resistance to individual therapeutic agents. Rifampin resistance in M. tuberculosis is largely associated with point mutations localized in a small core region of 81 base pairs (bp) in the rpoB gene, which encodes for the RNA polymerase beta subunit (The rpoB Gene of M. tuberculosis, Miller, et al., Antimicrobial Agents and Chemotherapy, 38, 805, 1994). Mutations in this 81-bp region of the gene (rpoB) account for rifampin resistance in 96% of M. tuberculosis strains and many other Mycobacterium species. The molecular mechanism of rifampin activity involves inhibition of DNA-dependent RNA polymerase. The clustering of the various rifampin resistance associated point mutations within the 81-bp region of rpoB, coupled with the high sequence conservation of this gene among strains of M. tuberculosis, form the basis for the current genetic tests for detection of mutations associated with rifampin resistance in M. tuberculosis. PCR primers specific for rpoB are used to amplify the portion of the rpoB gene that contains the most common mutations associated with rifampin resistance. The amplification products are then analyzed for the presence of rifampin-associated mutations. The most common methods used for mutation detection are DNA sequencing (Hunt, infra), DNA conformation-dependent methods such as single-strand conformation polymorphism (SSCP) (Talent et al. infra), or dideoxy fingerprinting (Felmee, et al., infra), as well as detection of specific PCR amplification products (Whelan, et al., infra).

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in M. tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance (Konno K. et al., Am. Rev. Respir. Pis. 1967, 98:461-469). The gene (pncA) encoding the M. tuberculosis PZase has recently been cloned and sequenced (Angelo S. and Ying Z., Nature Medicine, 1996, 2:662-667). Mutations in pncA gene are the major mechanism of pyrazinamide resistance in M. tuberculosis and these were identified in both acquired PZA-resistant M. tuberculosis and naturally resistant M. bovis strain (Angelo S. and Ying Z., supra; Angelo S. er a/., Antimicrobial Agents and Chemotherapy, 1997, 41:540-543; Srinand S. et al, Antimicrobial Agents and Chemotherapy, 1997, 41:636-640). Transformation of these strains with a functional pncA gene restored PZase activity and PZA susceptibility. This demonstrates that mutations identified in the pncA gene are the cause for PZA resistance in both types of resistant bacilli.

The current PZA susceptibility testing is performed in acidic medium (pH 5.5 to 6.0) as required for PZA activity, however M. tuberculosis strains grow poorly under this condition. The inhibitory effect of the medium required for testing for PZA resistance often leads to false determination of a resistant isolate as a susceptible one (JAMA, 1995, 273:916-919). A Bactec susceptibility test system for testing of PZA susceptibility of M. tuberculosis is commercially available. Insufficient growth in the test medium could lead to interpretable results (Miller M. A. et al., J Clin. Microbiology, 1995, 33:2468-2470). Another method for the determination of M. tuberculosis susceptibility to PZA utilizes determination of PZase activity (Wayne L. G., Am. Rev. Respir. Pis., 1974, 109:147-151 ; Trivedl S. S. and Desai S. G., Tubercle, 1987, 88:221-224). The automated and relatively faster Bactec method is preferred in the clinical laboratory; however, the growth based method requires at least 5 days for susceptibility testing following initial growth of the isolate. A rapid test method based on detection of pncA mutations, i.e., genotypic drug resistance determination, will not only avoid the problems of the current PZA susceptibility testing, but it will also open a new way for PZA susceptibility testing. Such an approach should prove to be useful for improving the treatment of disease and improving infection containment, especially in an era of increasing incidence of drug-resistant tuberculosis worldwide. In so far as multiple mutations that span the entire coding region of the pncA gene have been identified and shown to be associated with resistant phenotype, the molecular methods for the detection of any and all mutations are required for the genotypic detection of PZA resistance. Nucleic acid based methods for the detection of mutations in pncA gene for the determination of PZA susceptibility in M. tuberculosis, which are based on either automated sequencing or PCR-SSCP (single strand conformation polymorphism), were recently reported (Angelo S et al., Antimicrobial Agents and Chemotherapy, 1997, 41 , 540-543; U.S. Patent Application Serial No. 846,718 (Zhang Ying and Scorpio 1998).

Thus, it is desirable to have sensitive, simple and inexpensive methods for determining the drug resistance of microorganisms. The methods should minimize the number and complexity of steps and reagents.

2. Description of the Related Art.

A chip-based speciation and phenotypic characterization of microorganisms such as Mycobacterium tuberculosis using oligonucleotide sequences based on the rpoB gene is discussed in PCT application WO 97/29212 (Gingeras, et al.).

Detection of differences in nucleic acids is described in U.S. Patent Application Serial No. 08/771 ,623 (Lishanski, et al.).

U.S. Patent No. 5,643,723 (Persing, et al.) discloses detection of genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens.

U.S. Patent No. 5,470,723 (Walker, et al.) discloses detection of mycobacteria by multiplex nucleic acid amplification.

U.S. Patent No. 5,667,994 (Dilly, et al.) discloses amplification and detection of Mycobacterium avium complex species. Miller, et al., describe the rpoB gene of M. tuberculosis in Antimicrobial Agents and Chemotherapy (1994) 38:805.

Rapid and sensitive detection of antibiotic-resistant mycobacteria using oligonucleotide probe specific for ribosomal RNA precursors is discussed in U.S. Patent No. 5,770,373 (Britschgi, et al., 1) and U.S. Patent No. 5,726,021 (Britschgi, et al., 2) and U.S. Patent No. 5,712,095 (Britschgi, et al., 3).

U.S. Patent No. 5,652,106 (Plikaytis, et al.) discloses rapid amplification-based subtyping of mycobacterium tuberculosis.

Cockerill, et al., disclose detection of isoniazid resistant strains of M. tuberculosis in U.S. Patent Nos. 5,688,639 (Cockerill 1) and 5,658,733 (Cockerill 2).

Hunt, et al., disclose detection of genetic locus encoding resistance to rifampin in Mycobacterial cultures and clinical specimens in Diagn. Microbiol. Infect. Pis. (1994) 18:219.

Kapur, et al., disclose characterization by automated PNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase β subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas in J. Clinical Microbiol. (1994) 32(4): 1095-1098.

Telenti, et al., describe detection of rifampin resistance mutations in M. tuberculosis in Lancet (1993) 341 :647. Felmee, et a/., describe genotypic detection of rifampin resistance in M. tuberculosis: comparison of single-strand conformation polymorphism and dideoxy fingerprinting in J. Clinical Microbiology (1995) 33:1617.

Whelen, et al., disclose direct genotypic detection of rifampin resistance in M. tuberculosis in clinical specimens by using single-tube heminested PCR in J. Clinical Microbiology (1995) 33:556.

Williams, et al., discuss the evaluation of a polymerase chain reaction-based universal heteroduplex generator assay for direct detection of rifampin susceptibility of Mycobacterium tuberculosis from sputum specimens in CIP (1998) 26:446-450.

Telenti, et al., describe the direct automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis in Antimicrobial Agents and Chemotherapy (1993) 37(10):2054- 2058. The characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis is discussed by Scorpio, et al., Antimicrobial Agents and Chemotherapy (1997) 41:540-543.

Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the anti-tuberculosis drug pyrazinamide in tubercle bacillus is discussed by Scorpio, et al., Nature Medicine (1996) 2(6):662-667.

Hewlett, et al., (JAMA (1995) 273(12):916-917) describe drug-resistant tuberculosis and inconsistent results of pyrazinamide susceptibility testing.

Takahashi, et al., (Tuber Lung Pis (1997) 78(2): 117-122) (Medline Abstract) discuss mutation in pncA as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis.

Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide and comparison of Bactec method with pyrazinamidase assay is discussed by Miller, et al., in Journal of Clinical Microbiology (1995) 33(9):2468-2470. The pncA gene from naturally pyrazinamide-resistant Mycobacterium avium encodes pyrazinamidase and confers pyrazinamide susceptibility to resistant M. tuberculosis complex organisms is disclosed by Scorpio, et al., Microbiology (1997) 143:3367-3373 (Medline Abstract).

Scorpio, et al., Journal of Clinical Microbiology (1997) 35(1): 106-110 discloses rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene.

Identification of pyrazinamide-resistant mycobacteria and methods for treating mycobacterial infections is discussed in U.S. Patent No. 5,846,718 (Zhang).

U.S. Patent No. 5,786,149 (Leckie) discloses materials and methods for the detection of mycobacterium tuberculosis.

Perlin in U.S. Patent No. 5,876,933 discloses a method and system for genotyping.

U.S. Patent No. 5,554,503 (Down) discusses a sample processing method for Mycobacterium tuberculosis. U.S. Patent No. 5,376,527 (Robson) discloses a process for lysing mycobacteria.

Formation of a single base mismatch that impedes spontaneous DNA branch migration is described by Panyutin, et al., (1993) J. Mol. Biol., 230:413-424.

The kinetics of spontaneous DNA branch migration is discussed by Panyutin, et al., (1994) Proc. Natl. Acad. Sci. USA, 91 : 2021-2025. European Patent Application No. 0 450 370 A1 (Wetmur, et al.) discloses branch migration of nucleotides.

A displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U.S. Patent No. 4, 766,062 (Diamond, et al.). A strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.).

A method for the detection of the antibiotic resistance spectrum of mycobacterium species is disclosed in PCT application WO 95/33851 (De Beenhouwer, et al.).

PCT application WO/86/06412 (Fritsch, et al.) discusses process and nucleic acid construct for producing reagent complexes useful in determining target nucleotide sequences.

A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Patent No. 4,683,195.

SUMMARY OF THE INVENTION

One method in accordance with the present invention is directed to the detection of drug resistance in a strain of an organism. In the method the presence of at least one mutation in a predetermined region within the gene of the strain is detected. The predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the organism. To detect the mutation, a complex is formed comprising a first sequence representing the predetermined region of the gene of the organism and a second sequence representing the corresponding region of the gene of the wild type organism in double stranded form. Each member of at least one pair of non-complementary strands within the complex has a label. The association of the labels within the complex is detected wherein the association of the labels in the complex is related to the presence of the mutation. The presence of the mutation is related to the drug resistance of the strain.

Another aspect of the present invention is a method for detecting drug resistance in a strain of M. tuberculosis. The presence of at least one mutation in a first sequence representing a predetermined region within the gene of the strain is detected. A tailed target partial duplex A' is formed from the first sequence comprised of a duplex of the first sequence, a label at one end of the duplex, two non-complementary oligonucleotides, one linked to each strand. The tailed target partial duplex A' is provided in combination with a tailed reference partial duplex B' from a second sequence representing a corresponding region of a wild type strain having a label as a part thereof. The labels are present in non- complementary strands of the tailed target and tailed reference partial duplexes, respectively. The formation of a complex between the tailed partial duplexes is detected by means of the labels. The formation of the complex is directly related to the presence of the mutation. The presence of the mutation is related to the drug resistance of the strain of M. tuberculosis. Another aspect of the present invention is a method for detecting drug resistance in a strain of M. tuberculosis. The presence of at least one mutation in a first sequence representing a predetermined region within a gene of the strain is detected. An amplification of the first sequence is carried out by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA. One of the primers P1 and P2 comprises a label. The primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence. A primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A'. The primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement. A second sequence representing a region in wild type M. tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB. The primer P2 comprises a label when the primer P2 above comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label. The primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B'. The tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex B'. The binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected. The binding is directly related to the presence of the mutation. The presence of the mutation is related to the drug resistance of the strain of M. tuberculosis. Another aspect of the present invention is a method for detecting rifampin resistance in a strain of M. tuberculosis. A first sequence representing a predetermined region within the rpoB gene of the strain and a second sequence representing a sequence within wild type strain that corresponds to the first sequence are subjected to polymerase chain reaction. For each sequence there is employed a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO:1) and

5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO:2) and 5'-tailed primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3. The common nucleotide sequence is selected from the group consisting of

5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO: 17),

5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO: 18) and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO: 19).

Two different labels are employed for each labeled primer P2. The product of the polymerase chain reactions is a tailed partial duplex A' produced from the first sequence and a tailed partial duplex B' produced from the second sequence. The tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex B'. The binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected. The binding thereof is directly related to the presence of the mutation. The presence of the mutation is related to the rifampin resistance of the strain of M. tuberculosis.

Another embodiment of the present invention is a kit for carrying out a method for detecting rifampin resistance in a strain of M. tuberculosis. The kit comprises (a) a second sequence representing a sequence within wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2 selected from the group consisting of

5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO:1) and

5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO:2), wherein two different labels are employed for each labeled primer P2, and (c) 5'-tailed primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is selected from the group consisting of

5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO: 17), 5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO: 18), and

5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO: 19).

Another embodiment of the present invention is a method for detecting pyrazinamide resistance in a strain of M. tuberculosis. A medium is subjected to conditions for polymerase chain reaction. The medium comprises (i) a first sequence representing a predetermined region within the pncA gene of the strain, (ii) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3'

(SEQ ID NO:21), wherein two different labels are employed for each labeled primer P2, and (iii) 5'-tailed primers P1 and P3

5'-accatgctcgacattacgagtcaggagctgcaaaccaactc-3' (SEQ ID NO:22) 5'-gatcctaggcctcacgtatttcaggagctgcaaaccaactc-3' (SEQ ID NO 23) comprising a common nucleotide sequence, i e , a sequence that is the same in both P1 and P3, and a different oligonucleotide tail, such as set forth above, for each of the P1 and P3, wherein the common nucleotide sequence is 5'-tcaggagctgcaaaccaactc-3' (SEQ ID NO 24) (underlined above) The product of the polymerase chain reaction is a tailed target duplex A' produced from the first sequence A medium is subjected to polymerase chain reaction The medium comprises (i) a second sequence representing a sequence within wild type strain that corresponds to the first sequence, (n) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3' (SEQ ID NO 21), wherein two different labels are employed for each labeled primer P2, and (in) 5'- tailed primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3 wherein the common nucleotide sequence is 5'-t caggagctgcaaaccaactc-3' (SEQ ID NO 24) The product of the polymerase chain reaction is a tailed reference partial duplex B' produced from the second sequence The tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex B' The binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected The binding is directly related to the presence of the mutation The presence of the mutation is related to the pyrazinamide resistance of the strain of M tuberculosis Another embodiment of the present invention is a kit for carrying out a method for detecting pyrazinamide resistance in a first sequence representing a strain of M tuberculosis The kit comprises in packaged combination (a) a second sequence representing a sequence within wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3' (SEQ ID NO 21), wherein two different labels are employed for each labeled primer P2 and (c) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is 5'-t caggagctgcaaaccaactc-3' (SEQ ID NO 24) Another aspect of the present invention is a method for detecting the presence of a difference between two related nucleic acid sequences in an organism Amplification products are formed from both of the nucleic acid sequences using a medium comprising disrupted cells from the organism The amplification products are examined in the medium for the presence of a stable four stranded complex resulting from strand exchange. The presence of the difference in the nucleic acid sequences is determined based on the presence of the stable four stranded complex in the medium.

Another aspect of the present invention is a kit for detecting the presence of a difference between two related nucleic acid sequences in an organism. The kit comprises in packaged combination (a) two oligonucleotide primers each comprising a sequence that is hybridizable to the nucleic acid sequences in a region adjacent the site of potential difference and each comprising a label wherein the labels are different, and (b) a lysing agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1 and 1A are schematic diagrams depicting the formation and non-formation, respectively, of a quadramolecular complex in accordance with the present invention. Fig. 2 is a schematic diagram depicting an embodiment in accordance with the present invention. Fig. 3 is a schematic diagram depicting an initial amplification in accordance with the present invention.

Fig. 4 is a depiction of the sequence of the M. tuberculosis rpoB gene (GenBank Ace. No. U12205).

Fig. 5 is a graph of results obtained using a method in accordance with the present invention for the detection of an rpoB mutation in a M. tuberculosis strain versus a wild type M. tuberculosis.

Fig. 6 is a graph of results obtained using a method in accordance with the present invention for the detection of a an rpoB mutation in a M. tuberculosis strain versus a wild type M. tuberculosis employing cell disruption. Fig. 7 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain employing two different enzymes.

Fig. 8 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain. Fig. 9 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain and four mycobacteria other than tuberculosis strains. 11

Fig 10 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M tuberculosis strain versus a wild type M tuberculosis

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides a method and reagents for the determination of drug resistance in an organism such as a mycobacterium In general, the drug is one that is effective against the organism, usually, one that is employed in the treatment of an individual infected with the organism The method may be applied to any situation where drug resistance of an organism resides in the genome i e , where drug resistance is genetically determined The method usually involves PCR amplification for the production of substrates capable of formation of four-stranded DNA structures that will undergo spontaneous branch migration The formation of a four-stranded DNA structure or complex from DNA involves producing two partial duplexes by amplification by using three different primers in the polymerase chain reaction and allowing the amplified products to anneal The complex dissociates into normal duplex structures by strand exchange by means of branch migration when the hybridized portions of each partial duplex are identical However, where there is a difference between the two hybridized portions, the complex does not dissociate and can be detected as an indication of the presence of a mutation, which is related to the drug resistance of the organism under examination One aspect of the invention includes novel PCR primers for analysis of sequence alteration in the relevant region of rpoB gene of M tuberculosis Another aspect of the invention includes novel PCR primers for analysis of sequence alteration in the relevant region of pncA of M tuberculosis The methods of the invention are suitable for use in the clinical microbiology laboratory

Before proceeding further with a description of the specific embodiments of the present invention, a number of terms will be defined

Mycobacteria - a genus of bacteria that are acid-fast non-motile gram-positive rods The genus comprises several species that include, but are not limited to Mycobactenum afncanum, M avium, M bovis, M bovis-BCG, M cnelonae, M fortuitum, M gordonae, M mtracellulare, M kansasu, M microti, M scorfulaceum, M paratuberculosis and M tuberculosis Certain of these organisms are the causative agents of disease Nucleic acid -- a compound or composition that is a polymeric nucleotide or polynucleotide Generally speaking, nucleic acids include both nucleic acids and fragments thereof from any source in purified or unpuπfied form including DNA (dsDNA and ssDNA) and RNA, t-RNA, m-RNA, r-RNA, and the like In the context of the present invention, nucleic acids are derived from, or related to, the genes associated with a gene of a strain of a microorganism such as a bacteria, yeast, virus and the like The nucleic acid can be obtained from a biological sample by procedures well known in the art Where the nucleic acid is RNA, it is first converted to cDNA by means of a primer and reverse transcπptase The nucleotide polymerase used in the present invention for carrying out amplification and chain extension can have reverse transcπptase activity Sequences of interest may be embedded in sequences of any length of the chromosome, cDNA, plasmid, etc

Sample -- the material containing the nucleic acid Such samples include biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, and the like Other samples include clinical isolates, cell cultures and the like Accordingly, biological material may be pretreated with reagents to liquefy it and/or release nucleic acids from binding substances to thereby produce the sample to be tested or the biological may be used directly without release of nucleic acids Furthermore, the biological material such as cells from an organism may be disrupted by mechanical or chemical treatment either prior to or during the present method including amplification of the nucleic acid Mechanical treatment includes, for example, treatment with heat, sonication, treatment with microwaves and the like Chemical treatment includes, for example, treatment with a lysing agent Such pretreatments of samples and treatments for cell disruption are well known in the art Amplification of nucleic acids -- any method that results in the formation of one or more copies of a nucleic acid (exponential amplification) One such method for enzymatic amplification of specific sequences of DNA is known as the polymerase chain reaction (PCR), as described by Saiki, et al , supra This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence of the nucleic acid flanked by the primers The two different PCR primers are designed to anneal to opposite strands of the DNA at positions that allow the polymerase catalyzed extension product of one primer to serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers. Primer length can vary from about 10 to 50 or more nucleotides and are usually selected to be at least about 15 nucleotides to ensure high specificity. The double stranded fragment that is produced is called an "amplicon" and may vary in length form as few as about 30 nucleotides to 10,000 or more.

Chain extension of nucleic acids -- extension of the 3'-end of a polynucleotide in which additional nucleotides or bases are appended. Chain extension relevant to the present invention is template dependent, that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridized. The chain extension product sequence that is produced is complementary to the template sequence. Usually, chain extension is enzyme catalyzed, preferably, in the present invention, by a thermophilic DNA polymerase.

First sequence -- a sequence of nucleotides within a gene to be studied; a target nucleic acid sequence. The first sequence represents a predetermined region within the gene of a strain of an organism and may be double stranded or single stranded. When the sequence is single stranded, the method of the present invention produces a nucleic acid duplex comprising the single stranded first sequence.

The predetermined region exists within a portion of a gene of a strain of a microorganism to be studied. The predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the microorganism. In that regard the identity of the predetermined region is known at least to an extent sufficient so that such mutations are known. The identity of the predetermined region is generally known to allow preparation of various primers necessary for introducing one or more priming sites and/or conducting an amplification of the predetermined region in accordance with the present invention. Accordingly, other than the above, the identity of the gene may or may not be known. Examples of genes in conjunction with M. tuberculosis, by way of illustration and not limitation, include the rpoB gene and the pncA gene.

In general, in PCR, primers hybridize to, and are extended along (chain extended), at least a portion of a target nucleic acid sequence such as the predetermined region, and, thus, the predetermined region acts as a template. The minimum number of nucleotides in the target sequence is selected to assure that a determination of a mutation in accordance with the present invention can be achieved. Second sequence - a nucleic acid sequence that is related to the first sequence, a reference nucleic acid sequence In general, the second sequence represents a region of a wild type strain that corresponds to the predetermined region of the first sequence in that the two sequences are identical except for the presence of a mutation In certain situations the region of the wild type strain may be part of the sample Both the first and the second sequences are subjected to similar or the same amplification conditions As with the first sequence, the identity of the second sequence need be known so that the presence of mutations in the first sequence is known The identity of the corresponding region is generally known to allow preparation of various primers necessary for introducing one or more priming sites and/or conducting an amplification of the corresponding region in accordance with the present invention Accordingly, other than the above, the identity of the wild type strain may or may not be known The corresponding region of the wild type strain may be a reagent employed in the methods in accordance with the present invention In that regard the corresponding region may be obtained from a natural source or prepared by known methods such as those described below in the definition of ohgonucleotides

Holliday junction -- the branch point in a four-way junction in a complex of two identical nucleic acid sequences and their complementary sequences The junction is capable of undergoing branch migration resulting in dissociation into two double stranded sequences where sequence identity and complementarity extend to the ends of the strands

Complex -- a complex of four nucleic acid strands containing a Holliday junction, which is inhibited from dissociation into two double stranded sequences because of a mutation in one of the sequences and/or their complements Accordingly, the complex is quadramolecular

Mutation — a change in the sequence of nucleotides of a normally conserved nucleic acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence Mutations can generally be divided into two general classes, namely, base-pair substitutions and frameshift mutations The latter entail the insertion or deletion of one to several nucleotide pairs A difference of one nucleotide can be significant as to phenotypic normality or abnormality as in the case of, for example, sickle cell anemia

Partial duplex -- a fully complementary double stranded nucleic acid sequence wherein one end thereof has non-complementary oligonucleotide sequences, one linked to each strand of the double stranded molecule, each non-complementary sequence having 8 to 60, preferably, 10 to 50, more preferably, 15 to 40, nucleotides Thus, the partial duplex is said to be "tailed" because each strand of the duplex has a single stranded oligonucleotide chain linked thereto Duplex -- a double stranded nucleic acid sequence wherein all of the nucleotides therein are complementary

Oligonucleotide -- a single stranded polynucleotide, usually a synthetic polynucleotide The olιgonucleotιde(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides, and more preferably, 30 to 60 nucleotides in length

Various techniques can be employed for preparing an oligonucleotide utilized in the present invention Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis For short sequences (up to about 100 nucleotides) chemical synthesis will frequently be more economical as compared to the biological synthesis In addition to economy, chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step Furthermore, chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence The oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface This may offer advantages in washing and sample handling For longer sequences standard replication methods employed in molecular biology can be used such as the use of M 3 for single stranded DNA as described by J Messing (1983) Methods Enzvmol, 101. 20-78 Other methods of oligonucleotide synthesis include phosphotπester and phosphodiester methods (Narang, et al (1979) Meth Enzymol 68 90) and synthesis on a support (Beaucage, et al (1981) Tetrahedron Letters 22 1859-1862) as well as phosphoramidate technique, Caruthers, M H , et al_, "Methods in Enzymology," Vol 154, pp 287-314 (1988), and others described in "Synthesis and Applications of DNA and RNA," S A Narang, editor, Academic Press, New York, 1987, and the references contained therein

Oligonucleotide pπmer(s) -- an oligonucleotide that is usually employed in a chain extension on a polynucleotide template such as in, for example, an amplification of a nucleic acid The oligonucleotide primer is usually a synthetic oligonucleotide that is single stranded, containing a hybridizable sequence at its 3'-end that is capable of hybridizing with a defined sequence of the target or reference polynucleotide Normally, the hybridizable sequence of the oligonucleotide primer has at least 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence or primer binding site The number of nucleotides in the hybridizable sequence of an oligonucleotide primer should be such that stringency conditions used to hybridize the oligonucleotide primer will prevent excessive random non-specific hybridization Usually, the number of nucleotides in the hybridizable sequence of the oligonucleotide primer will be at least ten nucleotides, preferably at least 15 nucleotides and, preferably 20 to 50, nucleotides In addition, the primer may have a sequence at its 5'-end that does not hybridize to the target or reference polynucleotides that can have 1 to 60 nucleotides, preferably, 8 to 30 polynucleotides

Nucleoside tnphosphates -- nucleosides having a 5'-trιphosphate substituent The nucleosides are pentose sugar derivatives of nitrogenous bases of either puπne or pynmidine derivation, covalently bonded to the 1 '-carbon of the pentose sugar, which is usually a deoxyπbose or a πbose The puπne bases comprise adenιne(A), guanine (G), inosine (I), and derivatives and analogs thereof The pynmidine bases comprise cyto- sine (C), thymine (T), uracil (U), and derivatives and analogs thereof Nucleoside tnphosphates include deoxyπbonucleoside tnphosphates such as the four common tnphosphates dATP, dCTP, dGTP and dTTP and nbonucleoside tnphosphates such as the four common tnphosphates rATP, rCTP, rGTP and rUTP

The term "nucleoside tnphosphates" also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized and polymerized in a similar manner to the undeπvatized nucleoside tnphosphates Examples of such derivatives or analogs, by way of illustration and not limitation, are those which are biotinylated, amme modified, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like

Nucleotide - a base-sugar-phosphate combination that is the monomenc unit of nucleic acid polymers, i e , DNA and RNA Modified nucleotide - is the unit in a nucleic acid polymer that results from the incorporation of a modified nucleoside tπphosphate during an amplification reaction and therefore becomes part of the nucleic acid polymer

Nucleoside -- is a base-sugar combination or a nucleotide lacking a phosphate moiety Nucleotide polymerase - a catalyst, usually an enzyme, for forming an extension of a polynucleotide along a DNA or RNA template where the extension is complementary thereto. The nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside tnphosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template. Usually, the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 PNA polymerase, Klenow fragment, and reverse transcriptase, and are preferably thermally stable PNA polymerases such as Vent DNA polymerase, VentR DNA polymerase, Pfu DNA polymerase, Tag DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coli, plants, animals, virus, thermophilic bacteria, and so forth.

Wholly or partially sequentially -- when the sample and various agents utilized in the present invention are combined other than concomitantly (simultaneously), one or more may be combined with one or more of the remaining agents to form a subcombination. Subcombination and remaining agents can then be combined and can be subjected to the present method.

Hybridization (hybridizing) and binding—in the context of nucleotide sequences these terms are used interchangeably herein. The ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences. Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like.

Complementary -- two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence. Copy - means a sequence that is a direct identical copy of a single stranded polynucleotide sequence as differentiated from a sequence that is complementary to the sequence of such single stranded polynucleotide.

Conditions for extending a primer - includes a nucleotide polymerase, nucleoside tnphosphates or analogs thereof capable of acting as substrates for the polymerase and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium), pH, ionic strength, organic solvent (such as formamide), and the like.

Member of a specific binding pair ("sbp member") - one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as Hgand and receptor (antihgand). These may be members of an immunological pair such as antigen- antibody, or may be operator-repressor, nuclease-nucleotide, biotin-avidin, hormone- hormone receptor, IgG-protein A, DNA-DNA, DNA-RNA, and the like.

Ligand - any compound for which a receptor naturally exists or can be prepared. Receptor ("antihgand") - any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site. Illustrative receptors include naturally occurring and synthetic receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors, oligonucleotides, protein A, complement component C1q, or DNA binding proteins and the like.

Small organic molecule - a compound of molecular weight less than about 1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, digoxin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens, etc. The small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a support.

Support or surface - a porous or non-porous water insoluble material. The support can be hydrophihc or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed. Binding of sbp members to a support or surface may be accomplished by well- known techniques, commonly available in the literature See, for example, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J Biol Chem , 245 3059 (1970) The surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like

Label - a member of a signal producing system Labels include reporter molecules or reporter groups that can be detected directly by virtue of generating a signal, and specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule such as oligonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein, organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and antidigoxin antibodies, respectively, polypeptides, polysacchandes, and the like In general, any reporter molecule that is detectable can be used The reporter molecule can be isotopic or nonisotopic, usually non-isotopic, and can be a catalyst, such as an enzyme, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, hposome, cell, etc , which may or may not be further labeled with a dye, catalyst or other detectable group, and the like The reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as lummol, a terbium chelator such as N- (hydroxyethyl) ethylenediaminetπacetic acid that is capable of detection by delayed fluorescence, and the like

The label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system As mentioned above, a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence Alternatively, the reporter molecule can bind to a nucleotide sequence by being bound to an sbp member complementary to an sbp member that comprises a label bound to a nucleotide sequence Examples of particular labels or reporter molecules and their detection can be found in U S Patent No 5,508,178, the relevant disclosure of which is incorporated herein by reference Signal producing system - the signal producing system may have one or more components, at least one component being the label The signal producing system generates a signal that relates to the presence of a mutation between the first sequence and the second sequence The signal producing system includes all of the reagents required to produce a measurable signal When a reporter molecule is not conjugated to a nucleotide sequence, the reporter molecule is normally bound to an sbp member complementary to an sbp member that is bound to or part of a nucleotide sequence Other components of the signal producing system can include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, coenzymes, substances that react with enzymic products, enzymes and catalysts, and the like The signal producing system provides a signal detectable by external means, such as by use of electromagnetic radiation, electrochemical detection, desirably by spectrophotometnc detection The signal-producing system is described more fully in U S Patent No 5,508,178, the relevant disclosure of which is incorporated herein by reference

Ancillary materials - various ancillary materials will frequently be employed in the methods and assays carried out in accordance with the present invention For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components Frequently, in addition to these additives, proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like

As mentioned above, one aspect of the present invention concerns a method for detecting drug resistance in a strain of an organism In the method the presence of at least one mutation in a first sequence representing a predetermined region within the gene of the strain is detected The predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the organism This may be most readily understood with regard to drug resistant phenotypes that appear to arise as a consequence of chromosomal mutations In this situation multidrug resistance appears to be due to stepwise accumulation of mutations conferring resistance to individual therapeutic agents Accordingly, to detect drug resistance the predetermined region is analyzed to detect a mutation In this regard, a quadramolecular complex is formed comprising the first sequence and a second sequence that represents the corresponding region of the gene of the wild type organism in double stranded form Usually, the complex comprises a Holliday junction Each member of at least one pair of non-complementary strands within the complex has a label The association of the labels within the complex is detected wherein the association of the labels in the complex is related to the presence of the mutation The presence of the mutation is related to the drug resistance of the strain

This aspect of the present invention is depicted in Fig 1 A mutation M is present in a first sequence A The method comprises forming from the first sequence a tailed target partial duplex A' comprised of a duplex of the first sequence, a label L1 and at one end of the duplex, two non-complementary ohgonucleotides A1 and A2, one linked to each strand of duplex A' Ohgonucleotides A1 and A2 have from 8 to 60 nucleotides, preferably, 15 to 40 nucleotides The tailed target partial duplex is provided in combination with a labeled tailed reference partial duplex B' lacking mutation M, which is a second sequence of the wild type strain of the organism Accordingly, one terminus of the tailed reference partial duplex B' has, as the end part of each strand, a sequence of nucleotides B1 and B2, respectively, that are complementary to A2 and A1 , respectively, of A' and are not complementary to each other Labels L1 and L2 are present in non- complementary strands of the tailed target and tailed reference partial duplexes A' and B', respectively, where L1 and L2 may be the same or different

A complex C is formed Oligonucleotide tail A1 of A' is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B' Because oligonucleotide tails A1 and B1 are different, branch migration can only proceed away from these tails and then only until mutation M is reached, at which point branch migration stops Thus, when a mutation is present, complex C is stable and can be detected by determining whether both labels L1 and L2 have become associated The association of the labels indicates the presence of complex C The formation of complex C is directly related to the presence of the mutation If mutation M is not present in the nucleic acid (see Fig 1A), branch migration continues until complete strand exchange has occurred and only the separate duplexes D and E are present In this event no complex C is detected As mentioned above, the presence of the mutation is related to the drug resistance of the gene that is being tested

The invention may be further understood with reference to Fig 2, which depicts, by way of example and not limitation, the method of the present invention in step-wise fashion The production of tailed target partial duplex A' from target nucleic acid duplex A comprising the first sequence having mutation M and the production of tailed reference partial duplex B' from reference nucleic acid duplex B comprising the second sequence of the wild type strain are shown In Fig 2, A is amplified by the polymerase chain reaction using primers P1 and P2 to produce an amplicon AA Primer P2 contains a label L1 and primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence The amplification is carried out in the presence of a nucleotide polymerase and nucleoside tnphosphates using temperature cycling Amphcon AA has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P1 The unlabeled strand has a 5'-end portion B1 of primer P1 and the labeled strand has a corresponding 3'-end portion A2, which is the complement of B1

The above amplification is carried out by polymerase chain reaction (PCR) utilizing temperature cycling to achieve denaturation of duplexes, oligonucleotide primer annealing, and primer extension by thermophilic template dependent nucleotide polymerase In conducting PCR amplification of nucleic acids, the medium is cycled between two to three temperatures The temperatures for the present method for the amplification by PCR generally range from about 50°C to about 100°C, more usually, from about 60°C to about 95°C Relatively low temperatures of from about 50°C to about 80°C are employed for the hybridization steps, while denaturation is carried out at a temperature of from about 80°C to about 100°C and extension is carried out at a temperature of from about 70°C to about 80°C, usually about 72°C to about 74°C The amplification is conducted for a time sufficient to achieve a desired number of copies for an accurate determination of whether or not the first sequence has a mutation Generally, the time period for conducting the method is from about 10 seconds to about 10 minutes per cycle and any number of cycles can be used from about 1 to as high as about 60 or more, usually about 10 to about 50, frequently, about 20 to about 45 As a matter of convenience it is usually desirable to minimize the time period and the number of cycles In general, the time period for a given degree of amplification can be minimized, for example, by selecting concentrations of nucleoside tnphosphates sufficient to saturate the polynucleotide polymerase, by increasing the concentrations of polynucleotide polymerase and polynucleotide primer, and by using a reaction container that provides for rapid thermal equilibration Generally, the time period for conducting the amplification in the method of the invention is from about 5 to about 200 minutes In an example of a typical temperature cycling as may be employed, the medium is subjected to multiple temperature cycles of heating at about 90°C to about 100°C for about 10 seconds to about 3 minutes and cooling to about 65°C to about 80°C for a period of about 10 seconds to about 3 minutes Referring again to Fig 2, a chain extension of primer P3 along the labeled strand of amplicon AA then occurs to produce tailed target partial duplex A' Primer P3 is comprised of a 3'-end portion Pa, which is identical to Pa of primer P1 and which binds to the labeled strand of AA P3 has 5'-end portion A1 that is not complementary to amphcon AA The chain extension occurs in the presence of a nucleotide polymerase and nucleoside tnphosphates under appropriate temperature conditions so that only the complementary strand of the labeled strand is produced and not a copy In a step-wise approach this may be achieved by removing primers P2 and P1 prior to extension of P3 in a manner as described hereinbelow However, removal of primers P2 and P1 is not necessary and all of the reactions may be carried out in the same medium The complementary unlabeled strand of tailed target partial duplex A' has a 5'-end portion A1 , which is not complementary to the 3'-end portion A2 of the labeled strand of A' Unless the PCR reaction is carried out to produce an excess of the labeled strand, there will also be present the unlabeled strand from the amplification This strand is not a template during chain extension to form partial duplex A'

The conditions for carrying out the chain extension in accordance with the present invention are similar to those for the amplification described above In general, the medium is heated to a temperature of about 90°C to about 100°C for a period of about 5 to about 500 seconds and then cooled to about 20°C to about 80°C for a period of about 5 to about 2000 seconds followed by heating to about 40°C to about 80°C for a period of about 5 to about 2000 seconds Preferably, the medium is subjected to heating at about 90°C to about 100°C for a period of about 10 seconds to about 3 minutes, cooling to about 50°C to about 75°C for a period of about 10 seconds to about 2 minutes and heating to about 70°C to about 80°C for a period of about 30 seconds to about 5 minutes It is also within the purview of the present method to use only two temperature cycles, namely, denaturation at about 90°C to about 100°C for a period of about 10 seconds to about 3 minutes and cooling to about 65°C to about 85°C for a period of about 10 seconds to about 2 minutes

In carrying out the present method, an aqueous medium is employed The sample to be analyzed is present in the medium For example, the sample may be cells from the organism that have been disrupted as discussed above or the medium may be treated mechanically or chemically to disrupt the cells Other polar cosolvents may also be employed, usually oxygenated organic solvents of from 1-6 more usually from 1-4, carbon atoms, including alcohols, ethers and the like Usually these cosolvents, if used, are present in less than about 70 weight percent, more usually in less than about 30 weight percent

The pH for the medium is usually in the range of about 4 5 to about 9 5, more usually in the range of about 5 5 to about 8 5, and preferably in the range of about 6 to about 8, usually about 8 In general for amplification, the pH and temperature are chosen and varied, as the case may be, so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the oligonucleotide primer with the target nucleic acid sequence, extension of the primer, and dissociation of the extended primer Various buffers may be used to achieve the desired pH and maintain the pH during the determination Illustrative buffers include borate, phosphate, carbonate, Tπs, barbital and the like The particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another The buffer employed in the present methods normally contains magnesium ion (Mg²⁺) at a concentration of from about 1 to about 10 mM, which is commonly used with many known polymerases, although other metal ions such as manganese have also been used

Preferably, magnesium ion is used at a concentration of from about 1 to about 20mM, preferably, from about 1 to about 10mM The magnesium can be provided as a salt, for example, magnesium chloride and the like The primary consideration is that the metal ion permits the determination of a mutation in accordance with the present invention The concentration of the nucleotide polymerase is usually determined empirically

Preferably, a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over about 5-fold, preferably about 2-fold The primary limiting factor generally is the cost of the reagent The amount of the nucleic acid that is to be examined in accordance with the present invention can be as low as one or two molecules in a sample The priming specificity of the primers used for the detection of a mutation and other factors will be considered with regard to the need to conduct an initial amplification of the first sequence It is within the purview of the present invention for detection of a mutation to carry out a preliminary amplification reaction to increase, by a factor of about 10² or more, the number of molecules of the first sequence The amplification can be by any convenient method such as PCR, amplification by single primer, NASBA, and so forth, but will preferably be by PCR

The amount of the first sequence to be subjected to subsequent amplification using primers in accordance with the present invention may vary from about 1 to about 10 3 8 ₂1

10 , more usually from about 10 to about 10 molecules, preferably at least about 10- M

-10 -19 -1 in the medium and may be about 10 to about 10 M, more usually about 10 to about

-19

10 M

If an initial amplification of the first sequence is carried out to increase the number of molecules, it may be desirable, but not necessary, to remove, destroy or inactivate the primers used in the initial amplification depending on the nature of the protocol utilized Accordingly, when the present method is carried out using step-wise addition of reagents for each separate reaction, primer P1 should be removed prior to the extension of primer P3 On the other hand, for example, in the embodiment described hereinbelow where the reactions are carried out simultaneously, it is not necessary to remove any of the primers An example, by way of illustration and not limitation, of an approach to destroy the primers is to employ an enzyme that can digest only single stranded DNA For example, an enzyme may be employed that has both 5' to 3' and 3' to 5' exonuclease activities, such as, e g , exo VII The medium is incubated at a temperature and for a period of time sufficient to digest the primers Usually, incubation at about 20°C to about 40°C for a period of 10 to 60 minutes is sufficient for an enzyme having the above activity The medium is next treated to inactivate the enzyme which can be accomplished, for example, by heating for a period of time sufficient to achieve inactivation Inactivation of the enzyme can be realized usually upon heating the medium at about 90°C to about 100°C for about 0 5 to about 30 minutes Other methods of removing the primers will be suggested to those skilled in the art

The amount of the oligonucleotide pπmer(s) used in the amplification reaction in the present invention will be at least as great as the number of copies desired and will usually be about 10-9 to about 10-3 M, preferably, about 10-7 to about 10-4 M Prefer- ably, the concentration of the oligonucleotide ρπmer(s) is substantially in excess over, preferably at least about 100 times greater than, more preferably, at least about 1000 times greater than, the concentration of the first sequence The concentration of the nucleoside tnphosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension The nucleoside tnphosphates are usually present in about 10-6 to about 10-2M, preferably about 10-5 to about 10-3M

The order of combining the various reagents may vary The first sequence may be combined with a pre-prepared combination of primers nucleoside tnphosphates and nucleotide polymerase Alternatively, the target nucleic acid, for example, can be combined with certain primers together with the nucleoside tnphosphates and polymerase After temperature cycling is carried out, the reaction mixture can be combined with the remaining primers As depicted in Fig 2, second sequence B, in the presence of primer P2 and primer

P3, is amplified in a polymerase chain reaction to produce amplicon BB The amplification occurs using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside tnphosphates B is comprised of a sequence identical to A except for mutation M Generally, primer P2 used for this amplification contains a label L2 that may be the same as or different than L1 Amplicon BB has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P3 The unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1

A chain extension of pπmer P1 along the labeled strand of amphcon BB occurs, under the conditions mentioned above for the chain extension of primer P3 along the labeled strand in duplex AA, to produce tailed reference partial duplex B' comprising the second sequence As mentioned above, primer P1 is comprised of portion Pa, which binds to the labeled strand of BB and portion B1 that does not bind to amphcon BB The chain extension is carried out in the presence of a nucleotide polymerase and nucleoside tnphosphates under appropriate temperature conditions so that only the complement of the labeled strand is produced and not a copy The extended primer P1 has a 5'-end portion B1 , which is not complementary to end portion B2 of the labeled strand of B' As can be seen, A' and B' are related in that each of their labeled strands is complementary, except for mutation M, to the unlabeled strand of the other The above polymerase chain reactions for first sequence A and sequence B may be carried separately or they may be carried out in a combined reaction medium In the present invention it is preferable that the polymerase chain reactions be conducted separately However, under certain circumstances and for certain genes and organisms the reactions may be carried out in combination Where the polymerase chain reactions are conducted separately, the aliquots of the reaction mixtures are combined and subjected to conditions for branch migration Once combined, the strands of partial duplexes A' and B' bind and undergo branch migration Incubation is carried out at a temperature of about 30°C to about 75°C, preferably about 60°C to about 70°C, for at least one minute, preferably, about 20 to about 90 minutes, wherein complex C is formed Oligonucleotide tail A1 of A' is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B' Branch migration within complex C continues under the above temperature conditions with separation of the complex into duplexes D and E unless a mutation M is present, whereupon branch migration and strand dissociation is inhibited Complex C is then detected, the presence of which is directly related to the presence of mutation M, which in turn is directly related to drug resistance

In the embodiment depicted in Fig 2, labels L1 and L2 are incorporated into the partial duplexes that comprise complex C and provide a means for detection of complex C This is by way of illustration and not limitation and other convenient methods for detecting complex C may be employed, such as the use of a receptor for the complex In this approach there is required only one label, L1 or L2, which comprises an sbp member or a reporter molecule A receptor for the sbp member and a receptor that can bind to complex C by virtue of a feature other than L1 or L2 can both bind to complex C and provide a means for detection

As mentioned above, under certain circumstances the reactions of the present invention may be carried out in the same reaction medium and many or all of the reactions may be carried out simultaneously In this approach a combination is provided in a single medium The combination comprises (i) a sample containing a first sequence of within the gene of the strain of a microorganism where the region is suspected of having a mutation, (n) a second sequence of the wild type strain of the microorganism, which may be added separately if it is not known to be present in the sample, (in) a nucleotide polymerase, (iv) nucleoside tnphosphates, and (v) primers P1 , P2 and P3, wherein P2 may include primer P2 labeled with L1 and primer P2 labeled with L2, or P2 may be unlabeled and primers P1 and P3 may be labeled respectively with L1 and L2 The medium is then subjected to multiple temperature cycles of heating and cooling to simultaneously achieve all of the amplification and chain extension reactions as depicted in Fig 2 Preferably, each cycle includes heating the medium at about 90°C to about 100°C for about 10 seconds to about 3 minutes, cooling the medium to about 60°C to about 70°C for a period of about 10 seconds to about 3 minutes, and heating the medium at about 70°C to about 75°C for a period of about 10 seconds to about 3 minutes although different temperatures may be required depending on the lengths of the primer sequences Following the above temperature cycling the medium is subjected to heating for a period of time sufficient to denature double stranded molecules, preferably, at about 90°C to about 99°C for about 10 seconds to about 2 minutes, and cooled to about 40°C to about 80°C, preferably about 60°C to about 70°C, and held at this temperature for at least one minute, preferably for about 20 minutes to about 2 hours Following cooling of the medium all possible partial and complete duplexes are formed that can form from 1) single strands that have any combination of wild type or mutant sequences and 5'-ends A2 and B2, and 2) single strands having any combination of wild type or mutant sequences and 5'-ends A1 or B1 wherein the strands may further be labeled with either L1 or L2 when L1 and L2 are different Among the partial duplexes that are formed are the tailed partial duplexes A' and B', which can bind to each other to form complex C, which does not dissociate into duplexes D and E when a mutation is present A determination of the presence of such a complex is then made to establish the presence of a mutation in the first sequence of the gene When primers P1 and P3 are labeled instead of primer P2, the labels L1 and L2 in partial duplexes A' and B' are attached to tails A1 and B1 , respectively, which still provides for detection of complex C when a mutation is present

As mentioned above, it is preferable to conduct the polymerase chain reactions separately Thus, for example, PCR amplification of first sequence A and second sequence B, each using primers P1 , P2 and P3, can be conducted in separate solutions The solution can then be combined, heated to about 90°C to about 100°C to denature strands and then incubated as before at about 40°C to about 80°C to permit formation of duplexes and complex C when a mutation is present Detection of complex C can then be carried out directly in the combined solutions or by adding reagents required for detection or by separating the complex C, for example, on a solid surface, and detecting its presence on the surface

When a single reaction medium is used for carrying out the method in accordance with the present invention or the initial concentration of the sample DNA is low, it may be necessary to conduct an initial amplification to increase the concentration of the first sequence molecules and the second sequence molecules relative to that of other nucleic acids that may be present in the sample To this end such initial amplification can be carried out using two additional primers PX1 and PX2 that bind to sites on the target and reference nucleic acids, which sites are upstream of the P2 binding site and the P1 and P3 binding site, respectively This initial amplification can be carried out in the same medium as the above reactions Thus, primers PX1 , PX2, P1 , P2 and P3 may all be combined with the target and reference sequences prior to temperature cycling This is more readily seen in Fig 3, which depicts the initial amplification for a first sequence TS Two primers PX1 and PX2 are employed and bind to sites on TS that are upstream of the sites to which primers P1 and P2, respectively, bind These sites are indicated by Pa' and P2', respectively, in Fig 3 The sites to which primers PX1 and PX2 bind are generally within about 0 to about 500 nucleotides, preferably, about 0 to about 200 nucleotides away from Pa' and P2' and may overlap partially or completely with Pa' and P2' PX1 and PX2 are extended along their respective strands The amplification produces multiple copies of target nucleic acid sequence A After appropriate denaturing, primers P1 and P2 are allowed to anneal to and extend along the respective strands of A to produce multiple copies of AA The above also occurs for the second sequence B to produce multiple copies thereof, which is further amplified with primers P2 and P3 to produce multiple copies of BB

Preferably, when an initial amplification using primers PX1 and PX2 is carried out, these primers will be designed to anneal to the first sequence and the second sequence nucleic acids at a higher temperature than that for primers P1 , P2 and P3, respectively This is usually achieved by selecting PX1 and PX2 sequences that are longer or more GC rich than P2 and the Pa binding sequence in P1 and P3 The initial amplification is then carried out at temperatures that exceed the temperature required for binding P1 , P2 and P3 and the subsequent amplifications to form AA and BB are carried out at lower temperatures that permit P1 P2 and P3 to bind It is then possible to detect a mutation by combining the sequences, primers PX1 , PX2, P1 , P2 and P3 wherein P2 or P1 and P3 are labeled, polynucleotide polymerase, nucleotides tnphosphates, and optionally the reagents needed to detect complex C, all in one medium The initial amplification is carried out at temperatures that permit PX1 and PX2, but not P1 , P2 and P3, to bind to the target sequence whereupon sequences A and B are formed Temperature cycling is then carried out at a lower temperature where P1 , P2 and P3 can bind and be extended The mixture is then heated to about 90°C to about 100°C to denature the duplexes and cooled to permit formation of partial duplexes AA and BB and their hybridization to form complex C The complex can then be detected directly if all of the necessary reagents are present or detection can be carried out in a separate step The nature of primers PX1 and PX2, as well as the appropriate temperature for binding of these primers to the target sequence, are generally determined empirically with reference to the nucleotide composition of primers P1 , P2 and P3 In another approach in accordance with the present invention, priming sites for primers P1 , P2 and P3 may be introduced to the first sequence and the second sequence, usually flanking these regions To this end an initial amplification can be carried out using two additional primers PX1ι and PX2ι that bind to sites on the first sequence and the second sequence This initial amplification is usually carried out in a different reaction container from that in which the above reactions are carried out Thus, primers PX1 ι and PX2ι are combined with the first sequence and the second sequence in an appropriate medium and subjected to temperature cycling The primers PX1 ι and PX2ι bind to respective priming sites on first sequence TS PX1 ι has a 3'-end portion that can hybridize with the first sequence and 5'-end portion Pa that cannot hybridize with the first sequence PX2ι has a 3'-end portion that can hybridize with the first sequence and 5'-end portion P2 that cannot hybridize with the first sequence PX1 ι and PX2ι are extended along their respective strands The amplification produces multiple copies of extended primers that comprise the relevant portion of the first sequence flanked by priming sites Pa and P2, designated A The reaction products from this initial amplification are combined with primers P1 , P2 and P3 Primers P1 and P2 anneal to and extend along the respective strands of A to produce multiple copies of AA The above also occurs for the reference DNA to produce multiple copies of reference nucleic acid B, which is further amplified with primers P2 and P3 to produce multiple copies of BB The remainder of the reactions that occur are as described above to give A' and B', which then can form complex C

The above embodiment permits the use of universal primers P1 , P2 and P3 This means that one set of primers for carrying out the reactions to produce complex C can be used for a first sequence of a gene from any strain Such an approach involves the use of primers PX1 ι and PX2ι, which are designed to introduce to the target and reference sequences priming sites for universal primers P1 , P2 and P3 The relationship of PX11 and PX2ι are such that each contains a 5'-end portion that corresponds to the priming sequence portion at the 3'-end of primers P1 , P2 or P3 as the case may be In the embodiment shown in Fig 9, PX1 ι contains 5'-end portion P2, which results in the introduction of priming site P2' in TS to which P2 can hybridize

In the present invention one means of detecting the quadramolecular complex involves the use of two labels on non-complementary strands The labels become associated by virtue of both being present in the quadramolecular complex if a mutation is present Detection of the two labels in the complex provides for detection of the complex Generally, the association of the labels within the complex is detected This association may be detected in many ways For example, one of the labels can be an sbp member and a complementary sbp member is provided attached to a support Upon the binding of the complementary sbp members to one another, the complex becomes bound to the support and is separated from the reaction medium The other label employed is a reporter molecule that is then detected on the support The presence of the reporter molecule on the support indicates the presence of the complex on the support, which in turn indicates the presence of the mutation in the target nucleic acid sequence An example of a system as described above is the enzyme-linked immunosorbent assay (ELISA), a description of which is found in "Enzyme-lmmunoassay," Edward T Maggio, editor, CRC Press, Inc , Boca Raton, Florida ( 980) wherein, for example, the sbp member is biotin, the complementary sbp member is streptavidin and the reporter molecule is an enzyme such as alkaline phosphatase

Detection of the signal will depend upon the nature of the signal producing system utilized If the reporter molecule is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometπcally, or a product that can be detected by other spectrometπc or electrometπc means If the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined Where the label is a radioactive group, the medium can be counted to determine the radioactive count

The association of the labels within the complex may also be determined by using labels that provide a signal only if the labels become part of the complex This approach is particularly attractive when it is desired to conduct the present invention in a homogeneous manner Such systems include enzyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemilummescence assay, induced luminescence assay, latex agglutination and the like

In one aspect of the present invention detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand Other modes of binding to the particles are well within the skill of the art such as those mentioned in the examples herein Such a particle serves as a means of segregating the bound first sequence from the bulk solution, for example, by settling, electrophoretic separation or magnetic separation A second label, which becomes part of the complex if a mutation is present, is a part of the signal producing system that is separated or concentrated in a small region of the solution to facilitate detection Typical labels that may be used in this particular embodiment are fluo- rescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U S Serial No 07/923,069 filed July 31 , 1992, the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent labels

Preferably, the particle itself can serve as part of a signal producing system that can function without separation or segregation The second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method A variety of combinations of labels can be used for this purpose When all the reagents are added at the beginning of the reaction, the labels are limited to those that are stable to the elevated temperatures used for amplification, chain extension, and branch migration In that regard it is desirable to employ as labels polynucleotide or polynucleotide analogs having about 5 to about 20 or more nucleotides depending on the nucleotides used and the nature of the analog Polynucleotide analogs include structures such as polyπbonucleotides, polynucleoside phosphonates, peptido-nucleic acids, polynucleoside phosphorothioates, homo DNA and the like In general, unchanged nucleic acid analogs provide stronger binding and shorter sequences can be used Included in the reaction medium are oligonucleotide or polynucleotide analogs that have sequences of nucleotides that are complementary One of these oligonucleotides or oligonucleotide analogs is attached to, for example, a reporter molecule or a particle The other is attached to a primer, either primer P2 or primer P1 and/or P3 as a label Neither the oligonucleotide nor polynucleotide analog should serve as a polynucleotide polymerase template This is achieved by using either a polynucleotide analog or a polynucleotide that is connected to the primer by an abasic group The abasic group comprises a chain of 1 to about 20 or more atoms, preferably at least about 6 atoms, more preferably, about 6 to about 12 atoms such as, for example, carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus, which may be present as various groups such as polymethylenes, polymethylene ethers, hydroxylated polymethylenes, and so forth The abasic group conveniently may be introduced into the primer during solid phase synthesis by standard methods

Under the proper annealing temperature an oligonucleotide or polynucleotide analog attached to a reporter molecule or particle can bind to its complementary polynucleotide analog or oligonucleotide separated by an abasic site that has become incorporated into partial duplexes A' and B' as labels during amplification If the partial duplexes become part of a quadramolecular complex, the reporter molecule or particle becomes part of the complex By using different polynucleotide analogs or oligonucleotide sequences for labels, L1 and L2, two different reporter molecules or particles can become part of the complex Various combinations of particles and reporter molecules can be used

The particles, for example, may be simple latex particles or may be particles comprising a sensitizer, chemiluminescer, fluorescer, dye, and the like Typical parti- cle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a tπtiated reporter molecule and a particle containing a scintillator Typical reporter molecule pairs include a fluorescent energy donor and a fluorescent acceptor dye Typical particle pairs include (1) two latex particles, the association of which is detected by light scattering or turbidimetry, (2) one particle capable of absorbing light and a second label particle which fluoresces upon accepting energy from the first, and (3) one particle incorporating a sensitizer and a second particle incorporating a chemiluminescer as described for the induced luminescence immunoassay referred to in U S Serial No 07/704,569, filed May 22, 1991 , entitled "Assay Method Utilizing Induced Luminescence" (and corresponding European Patent Application 0 515 194 A2), which disclosure is incorporated herein by reference

Briefly, detection of the quadramolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity The activated chemiluminescent compound subsequently produces light The amount of light produced is related to the amount of the complex formed

By way of illustration as applied to the present invention, a particle is employed, which comprises the chemiluminescent compound associated therewith such as by incorporation therein or attachment thereto The particles have a recognition sequence, usually an oligonucleotide or polynucleotide analog, attached thereto with a complementary sequence incorporated into one of the nucleic acid strands as a label, L1 Another particle is employed that has the photosensitizer associated therewith These particles have a recognition sequence attached thereto, which is different than that attached to the chemiluminescent particles A complementary sequence is incorporated as a label L2 in the nucleic acid strand in complex C that is not complementary to the nucleic acid strand carrying label L1 Once the medium has been treated in accordance with the present invention to form a quadramolecular complex C, the medium is irradiated with light to excite the photosensitizer, which is capable in its excited state of activating oxygen to a singlet state Because the chemiluminescent compound of one of the sets of particles is now in close proximity to the photosensitizer by virtue of the presence of the target polynucleotide having a mutation, the chemiluminescent compound is activated by the singlet oxygen and emits luminescence The medium is then examined for the presence and/or the amount of luminescence or light emitted, the presence thereof being related to the presence of quadramolecular complex C The presence of the latter indicates the presence of a mutation, which is related to the drug resistance of the gene under examination A particular application of the method of the present invention, by way of illustration and not limitation is a method for detecting rifampin resistance in a strain of M tuberculosis The presence of at least one mutation in a first sequence representing a predetermined region within the rpoB gene (see Fig 4) of the strain is detected The predetermined region is selected from the gene and is generally of a length such that it comprises a mutation cluster, i e , a portion of the gene that includes mutations that have been identified for the particular gene A preferred predetermined region for the rpoB gene includes, by way of example, the sequence represented by bp 1779 to bp 2097 of the rpoB gene (see Fig 4) It is within the purview of the present invention that the first sequence includes nucleotides on either side of the aforementioned predetermined regions The length of the first sequence is not critical and, usually, may be from about 10 to about 1000 nucleotides in length, more usually, from about 50 to about 600 nucleotides in length

An amplification of the first sequence is carried out by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA The nature of the primers is determined by the nature of the first sequence and in particular the nature of the predetermined region Exemplary P1 (and P3) primers include, by way of illustration and not limitation, the following

5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO 17) 5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO 18), and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO:19) wherein each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group NNA wherein each N is independently a modified nucleotide. One of the primers P1 , P2 and P3 comprises a label. The primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence. B1 does not hybridize with the first sequence and is referred to above as an oligonucleotide tail. By way of example and not limitation, for this aspect of the invention the oligonucleotide tail B1 may be selected from the following:

5'-ACCATGCTCGAGATTACGGAG-3' (SEQ ID NO:9) and

5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO: 10). Other oligonucleotide tails may be used. The main criteria for a tail useful in the present invention are that the tail does not hybridize with the genome under the conditions of the interrogation and that the tail has a high Tm for hybridization at the temperature of branch migration.

A primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A'. The primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement. The nature of Pa and A1 is determined by the nature of the first sequence and of B1. In general, A1 and B1 are selected to have minimal homology to the target sequence and to each other.

Exemplary P2 primers include by way of illustration and not limitation the following (where L = label): 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO:1) and 5'-L- CCACCCAGGACGTGGAGGC-3' (SEQ ID NO:2) wherein each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group NNT wherein each N is independently a modified nucleotide.

A1 does not hybridize with the first sequence, or with B1 , and is referred to above as an oligonucleotide tail. For this aspect of the invention and consistent with the nature of B1 , the oligonucleotide tail A1 may be selected from the following: 5'- ACCATGCTCGAGATTACGGAG-3' (SEQ ID NO:9) and

5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO: 10), which are set forth above in the discussion of B1. A second sequence representing a region in wild type M tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB Obviously, the nature of the second sequence is directly related to the nature of the first sequence The primer P2 used for the second sequence comprises a label when the primer P2 referred to above with respect to the first sequence comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label The primer P1 is extended by chain extension along one strand of amp con BB to produce a tailed reference partial duplex B' The tailed target partial duplex A' binds to the tailed reference partial duplex B' The binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected The binding is directly related to the presence of the mutation The presence of the mutation is related to the rifampin resistance of the strain of M tuberculosis

In the above manner sequence alteration in a sample polynucleotide that is representative of a mutation cluster is determined relative to a sequence that corresponds to the polynucleotide except for the mutation This alteration is then related to the drug resistance characteristics of the organism from which the sample polynucleotide was obtained In general, low signals obtained in the above process where induced luminescence is employed in detection indicate the identity of the sample polynucleotide relative to the reference sequence

Another embodiment of a method in accordance with the present invention, by way of illustration and not limitation, is a method for detecting pyrazinamide resistance in a strain of M tuberculosis The presence of at least one mutation in a first sequence representing a predetermined region within the pncA gene (see Example 4) of the strain is detected An amplification of the first sequence is carried out by polymerase chain reaction as described above for the detection of rifampin resistance, using the following as primers P1 and P3

5'-accatgctcgacattacgagtcaggagctgcaaaccaactc-3' (SEQ ID NO 22) 5'-gatcctaggcctcacgtatttcaggagctgcaaaccaactc-3' (SEQ ID NO 23) wherein each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group nna wherein each "n" is independently a modified nucleotide

As mentioned above, one of the primers P1 , P2 and P3 comprises a label In this particular embodiment P2 comprises a label The primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence B1 does not hybridize with the first sequence and is referred to above as an oligonucleotide tail By way of example and not limitation, for this aspect of the invention the oligonucleotide tail B1 may be selected from the following 5'-accatgctcgagattacgag-3' (SEQ ID NO 9) 5'-gatcctaggcctcacgtatt-3' (SEQ ID NO 10)

A primer P3 is extended by chain extension along one strand of amphcon AA to produce a tailed target partial duplex A' The primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement The nature of Pa and A1 is determined by the nature of the first sequence and of B1 In general, A1 and B1 are selected to have minimal homology to the target sequence and to each other A1 does not hybridize with the first sequence, or with B1 , and is referred to above as an oligonucleotide tail For this aspect of the invention and consistent with the nature of B1 , the oligonucleotide tail A1 may be selected from the following 5'-accatgctcgagattacgag-3' (SEQ ID NO 9) and 5'-gatcctaggcctcacgtatt-3' (SEQ ID NO 10), which are set forth above

Exemplary P2 primers include by way of illustration and not limitation the following (where L = label) 5'- L-tggtgccgcgtcggtagg-3' (SEQ ID NO 21) wherein the above sequence may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group nnt wherein each "n" is independently a modified nucleotide

A second sequence representing a region in wild type M tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amphcon BB Obviously, the nature of the second sequence is directly related to the nature of the first sequence The primer P2 used for the second sequence comprises a label when the primer P2 referred to above with respect to the first sequence comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label The primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B' The tailed target partial duplex A' binds to the tailed reference partial duplex B' The binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected The binding is directly related to the presence of the mutation The presence of the mutation is related to the pyrazinamide resistance of the strain of M tuberculosis As a matter of convenience, predetermined amounts of reagents employed in the present invention for the determination of a first sequence representing a predetermined region within the rpoB gene of the strain can be provided in a kit in packaged combination A kit can comprise in packaged combination (a) a second sequence representing a sequence within a wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO 1) and 5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO 2), wherein two different labels are employed for each labeled primer P2 and wherein each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group NNT wherein each N is independently a modified nucleotide, and (c) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence, i e , a sequence that is the same in both P1 and P3, and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is selected from the group consisting of 5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO 17), 5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO 18) and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO 19) wherein each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group NNA wherein each N is independently a modified nucleotide

Preferably, primer P2 can be labeled, but primers P1 and P3 alternatively may be labeled The kit can also include reagents for conducting an amplification of the first sequence prior to subjecting the first sequence to the methods of the present invention The kit can also include nucleoside tnphosphates and a nucleotide polymerase The kit can further include two additional oligonucleotide primers PX1 and PX2 where the primers are related in that a product of the extension of one along the first sequence serves as a template for the extension of the other The kit can further include particles as described above capable of binding to the label on at least one of the primers The kit can further include members of a signal producing system and also various buffered media, some of which may contain one or more of the above reagents Preferably, primers PX1 , PX2, P1 , P2 and P3 are packaged in a single container More preferably, at least all of the above components other than buffer are packaged in a single container In one aspect, the different oligonucleotide tails are 5'-ACCATGCTCGAGATTACGGAG-3' (SEQ ID NO 9) and 5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO 10) In another aspect the label is independently selected from the group consisting of ohgonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces In a particular embodiment the labels are biotin and digoxigenin

Another embodiment of a kit in accordance with the present invention is useful for the determination of pyrazinamide resistant M tuberculosis A kit for this embodiment can comprise in packaged combination (a) a second sequence representing a sequence within a wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3' (SEQ ID NO 21) wherein two different labels are employed for each labeled primer P2 as discussed above and wherein the above sequence may further comprise at the 3 -end one or more nucleotides and/or modified nucleotides such as, for example, the group nnt wherein each "n" is independently a modified nucleotide, and (c) 5'-taιled primers P1 and P3 5'-accatgctcgacattacgagtcaggagctgcaaaccaactc-3' (SEQ ID NO 22) 5'-gatcctaggcctcacgtatttcaggagctgcaaaccaactc-3' (SEQ ID NO 23) comprising a common nucleotide sequence, i e , a sequence that is the same in both P1 and P3, and a different oligonucleotide tail, such as set forth above, for each of the P1 and P3, wherein the common nucleotide sequence is 5'-t caggagctgcaaaccaactc-3' (SEQ ID NO 24), wherein the above sequence may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group nna wherein each "n" is independently a modified nucleotide Preferably, primer P2 can be labeled, but primers P1 and P3 alternatively may be labeled The kit can also include additional reagents such as set forth above in the kit for detection of rifampin resistant microorganism

Another embodiment of a kit is for detecting the presence of a difference between two related nucleic acid sequences in an organism The kit comprises in packaged combination (a) two oligonucleotide primers each comprising a sequence that is hybridizable to the nucleic acid sequences in a region adjacent the site of potential difference and each comprising a label wherein the labels are different, and (b) a lysing agent

The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the method in detecting a mutation Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including ex pients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method in accordance with the present invention Each reagent can be packaged in separate containers or some or all of the reagents can be combined in one container where cross-reactivity and shelf life permit In a particular embodiment of a kit in accordance with the present invention, the reagents are packaged in a single container The kits may also include a written description of a method in accordance with the present invention as described above

EXAMPLES

The invention is demonstrated further by the following illustrative examples Temperatures are in degrees centigrade (°C) and parts and percentages are by weight, unless otherwise indicated Melting temperatures and priming efficiency for individual ohgonucleotides were determined by computer analysis using the program OLIGO

The following definitions and abbreviations are used herein

Tπs - Tπs(hydroxymethyl)amιnomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD BSA - bovine serum albumin from Gibco BRL, Gaithersburg MD bp - base pairs wt (+) - wild type allele mut (-) - mutant allele sec - seconds hr - hours mm - minutes

Buffer A - 10mM Tπs-HCI (pH8 3), 50mM KCI, 1 5 μM MgCI₂ 200 μg/ml BSA

Buffer B - 100mM Tπs-HCI (pH8 3), 500mM KCI, 15mM MgCI₂ 200 μg/ml BSA

Buffer C - 0 1 M Tπs, 0 3M NaCI, 25 mM EDTA, 0 1 % BSA, 0 1 % dextran T-500, a 1 320 dilution of mouse IgG (HBR-1 from Scantibodies Laboratory Inc , Los Angeles, CA), 0 05% Kathon (Rohm and Haas, Philadelphia, PA), and 0 01 % gentamycin sulfate Buffer D - 10 mM Tπs-HCI, pH 8 3, 50 mM KCI, 1 5 mM MgCI₂, and 200 μg/ml

BSA). Buffer E - 100 mM Tπs-HCI, pH 8 3, 500 mM KCI, 15 mM MgCI₂, and 2 mg/ml

BSA) RLU - relative light units nt - nucleotides

MAD - maleimidylaminodextran

Ab - antibody

SAv - streptavidin MOPS - 3-(N-morρhohno)propane sulfonic acid sulfo-SMCC - sulfosuccinimidyl 4-(N-maleιmιdomethyl)cyclohexane-1-carboxylate

NHS - N-hydroxysuccinimide

EDAC - 1-ethyl-3-(3-dιmethylamιnopropyl)carbodιιmιde hydrochloπde

DMSO - dimethylsulfoxide MES - morphohnoethanesulfonate rpm - rotations per mm

EDTA - ethylenediaminetetraacetic acid

SATA - N-succinimidyl S-acetylthioacetate eq - equivalents A₂₈o - absorbance at wavelength 280 nanometers

DPP - 4,7-dιphenylphenanthrolιne

Eu(TTA)₃ - europium tπ-3-(2-thιenoyl)-1 ,1 ,1-tπfluoroacetonate

L or I - liter exo VII - exonuclease VII from E coli (from Amersham Life Science) (USB) DMF - dimethyl formamide

THF - tetrahydrofuran

MS - mass spectroscopy

NMR - nuclear magnetic resonance spectroscopy

TMSCI - tetramethylsilylchloπde ELISA - enzyme linked immunosorbent assay as described in "Enzyme- lmmunoassay," Edward T Maggio, CRC Press, Inc , Boca Raton, Florida (1980) dNTPs - 2'-deoxynucleosιde 5'-tπphosphates cloned Pfu - cloned Pyrococcus fuπosus DNA polymerase

M tb - Mycobacterium tuberculosis MOTT - Mycobacteria Other Than Tuberculosis PZA - pyrazinamide Materials-

All oligodeoxyπbonucleotides (PCR primers) and modified oligodeoxyπbonucleotides were obtained from Oligo Etc , Inc (Wilsonville, OR) Molecular weight size markers (50 to 2 kb) were purchased from Bio-Rad (Hercules, CA) Cloned Pfu polymerase and Turbo Pfu were purchased from Stratagene ( La Jolla, CA) 2'- deoxynucleoside 5'-tπphosphates (dNTPs) was purchased as 100 mM solution from Pharmacia Biotech (Piscataway, NJ) Bovine Serum Albumin (BSA) was purchased from Gibco LifeTech (Gaithersburg, MD) RNAse/DNAse-free water from Ambion (Austin, TX) PCR tubes were purchased from Corning and ISC Bioexpress (Kaysville, UT) Native 4- 10% precast gradient polyacrylamide gels were purchased from Novex and used to analyze the amplification products (Los Angeles, CA)

Monoclonal antibodies were produced by standard hybrid cell technology Briefly, the appropriated immunogen was injected into a host, usually a mouse or other suitable animal, and after a suitable period of time the spleen cells from the host were obtained Alternatively, unsensitized cells from the host were isolated and directly sensitized with the immunogen in vitro Hybrid cells were formed by fusing the above cells with an appropriate myeloma cell line and cultuπng the fused cells The antibodies produced by the cultured hybrid cells were screened for their binding affinity to the particular antigen, dig-BSA conjugate A number of screening techniques were employed such as, for example, ELISA screens Selected fusions were then recloned

Beads Acc-AbQig - Acceptor beads coupled (MAD) to the anti-Dig antibody

(with 377 antibody molecules per bead) were prepared as follows

Hydroxypropylaminodextran (1 NH₂/ 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala, Sweden) (50 g) in 150 L of H₂0 in a 3-neck round- bottom flask equipped with mechanical stirrer and dropping funnel To the above solution was added 18 8 g of Zn(BF₄)₂ and the temperature was brought to 87°C with a hot water bath Epichlorohydπn (350 mL) was added dropwise with stirring over about 30 mm while the temperature was maintained at 87-88°C The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C, then the mixture was cooled to room temperature Chlorodextran product was precipitated by pouring slowly into 3 L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven

The chlorodextran product was dissolved in 200 mL of water and added to 2 L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190 mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2 L of rapidly stirring methanol The final product was recovered by filtration and dried under vacuum

Hydroxypropylaminodextran (1 NH₂ / 7 glucose), prepared above, was dissolved in 50 mM MOPS, pH 7 2, at 12 5 mg/mL The solution was stirred for 8 hr at room temperature, stored under refrigeration and centrifuged for 45 mm at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material To 10 mL of this solution was added 23 1mg of Sulfo-SMCC in 1 mL of water This mixture was incubated for 1 hr at room temperature and used without further purification C-28 thioxene was prepared as follows

To a solution of 4-bromoanιlιne (30 g, 174 mmol) in dry DMF (200 mL) was added 1- bromotetradecane (89 3 mL, 366 mmol) and N,N-dιιsopropylethylamιne (62 2 mL, 357 mmol) The reaction solution was heated at 90°C for 16 hr under argon before being cooled to room temperature To this reaction solution was again added 1- bromotetradecane (45 mL, 184 mmol) and N,N-dιιsoproρylethylamιne (31 mL, 178 mmol) and the reaction mixture was heated at 90°C for another 15 hr After cooling, the reaction solution was concentrated in vacuo and the residue was diluted with CH₂CI₂ (400mL) The CH₂CI₂ solution was washed with 1 N aqueous NaOH (2x), H₂0, and brine, was dried over Na₂S0₄ and was concentrated in vacuo to yield a dark brown oil (about 110 g) Preparative column chromatography on silica gel by a Waters 500 Prep LC system eluting with hexane afforded a yellow oil that contained mainly the product (4-bromo-N,N-dι- (Cι₄H₂₉)-anιlιne) along with a minor component 1 -bromotetradecane The latter compound was removed from the mixture by vacuum distillation (bp 105-110°C, 0 6 mm) to leave 50 2 g (51%) of the product as a brown oil To a mixture of magnesium turnings (9 60 g, 395 mmol) in dry THF (30 mL) under argon was added dropwise a solution of the above substituted aniline product (44 7 g 79 mmol) in THF (250 mL) A few crystals of iodine were added to initiate the formation of the Gπgnard reagent When the reaction mixture became warm and began to reflux, the addition rate was regulated to maintain a gentle reflux After addition was complete, the mixture was heated at reflux for an additional hour The cooled supernatant solution was transferred via cannula to an addition funnel and added dropwise (over 2 5 hr) to a solution of phenylglyoxal (11 7 g, 87 mmol) in THF (300 mL) at -30°C under argon The reaction mixture was gradually warmed to 0°C over 1 hr and stirred for another 30 mm The resulting mixture was poured into a mixture of ice water (800 mL) and ethyl acetate (250 mL) The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3x) The combined organic phases were washed with H₂0 (2x), then brine and were dried over MgS0 Evaporation of the solvent gave 48 8g of the crude product as a dark green oily liquid Flash column chromatography of this liquid (gradient elution with hexane, 1 5 98 5, 3 97, 5 95 ethyl acetate hexane) afforded 24 7 g (50%) of the benzoin product (MS

(C₄₂H₆₉N0₂) [M-H]⁺ 618 6, ¹H NMR (250 MHz, CDCI₃) was consistent with the expected benzoin product To a solution of the benzoin product from above (24 7 g, 40 mmol) in dry toluene (500 mL) was added sequentially 2-mercaptoethanol (25 g, 320 mmol) and TMSCI (100 mL, 788 mmol) The reaction solution was heated at reflux for 23 hr under argon before being cooled to room temperature To this was added additional TMSCI (50 mL, 394 mmol), and the reaction solution was heated at reflux for another 3 hr The resulting solution was cooled, was made basic with cold 2 5 N aqueous NaOH and was extracted with CH₂CI₂ (3x) The combined organic layers were washed with saturated aqueous NaHC0₃ (2x) and brine, was dried over Na₂S0 and was concentrated in vacuo to give a brown oily liquid Preparative column chromatography on silica gel by using a Waters 500 Prep LC system (gradient elution with hexane, 1 99, 2 98 ethyl acetate hexane) provided 15 5 g (60%) of the C-28 thioxene as an orange-yellow oil (MS (C₄₄H₇₁NOS) [M-H]⁺ 661 6, ¹H NMR (250 MHz, CDCI₃) was consistent with the expected C-28 thioxene product 2-(4-(N,N-dι-(Cι₄H₂9)-anιlιno)-3-phenyl thioxene Carboxyl chemiluminescer (acceptor) beads (TAR beads)

The following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6% 1-chloro-9,10-bιs(phenylethynyl)anthracene (1-CI-BPEA) (from Aldπch Chemical Company) and 2 7% rubrene (from Aldπch Chemical Company) The particles were latex particles (Seradyn Particle Technology, Indianapolis IN) The dye composition (240-250 mM C-28 thioxene, 8-16 mM 1-CI-BPEA, and 20-30 mM rubrene) was incorporated into the latex beads in a manner similar to that described in U S Patent 5,340,716 issued August 23, 1994 (the 716 patent), at column 48, lines 24-45, which is incorporated herein by reference The dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65 4%), 2-ethoxyethanol (32 2%) and 0 1 N NaOH (2 3%) The beads were mixed and heated for 40 mm at 95°C with continuous stirring While the beads are being heated, the three chemiluminescent dyes were dissolved in 2-ethoxyethanol by heating them to 95°C for 30 mm with continuous stirring At the end of both incubations, the dye solution was poured into the bead suspension and the resulting mixture was incubated for an additional 20 mm with continuous stirring Following the 20-mιnute incubation the beads were removed form the oil bath and are allowed to cool to 40°C ± 10°C The beads were then passed through a 43-mιcron mesh polyester filter and washed The dyed particles were washed using a Microgon (Microgon Inc , Laguna Hills, CA) The beads were first washed with a solvent mixture composed of ethylene glycol and 2-ethoxyethanol (70%/30%) The beads were washed with 500 ml of solvent mixture per gram of beads This is followed by a 10 % aqueous ethanol (pH 10-11) wash The wash volume was 400 mL per gram of beads The beads were then collected and tested for % solid, dye content, particle size, signal and background generation Carboxyl acceptor beads prepared above (99 mg in 4 5 mL water) were added slowly with vortexing to 5 5 mL of MAD ammodextran from above, followed by 1 mL of 200 mg/mL NHS in 50 mM MES, pH 6, 1 mL of 200 mg/mL EDAC in water, and 450 μL of 1 M HCI, final pH 6 The mixture was incubated overnight at room temperature in the dark, then reacted with 200 mg succinic anhydride in 0 5 mL of DMSO for 30 mm at room temperature Freshly opened Surfact-Amps Tween-20 (Pierce Chemical Company,

Rockford, Illinois) was added and the beads were centrifuged 30 mm at 15,000 rpm in a Sorvall RC-5B centrifuge, washed by centπfugation with three 10mL portions of 50 mM MOPS, 50 mM EDTA, 0 1 % Surfact-Amps Tween-20 (Pierce Chemical Company), pH 7 2, and resuspended in 3 mL of the same Monoclonal anti-digoxm Ab (prepared as described above) was purified by ABx resin (Baker Chemical Company, Philhpsburg, NJ) and was dialyzed into 0 15 M NaCI, 5 mM Na₂HP0₄, pH 7 4 The anti-digoxm Ab was thiolated by mixing 622 μL (4 28 mg) with 10 2 μL of SATA (1 25 mg/mL in ethanol, 2 eq ), incubating for 1 hr at room temperature and dialyzing cold against 2x2 L of 150 mM NaCI, 10mM Na₂HP0₄, 1 mM EDTA, pH7 The thioacetylated antibody was deacetylated by adding 62 2 μL of hydroxylamme (1 M H₂NOH, 50 mM MOPS, 25 mM EDTA, pH 7), bubbling with argon and incubating for 1 hr at room temperature The product was applied to a Pharmacia PD-10 column (G-25) and eluted with 50 mM MOPS, 50 mM EDTA, pH 7 2, bubbled with argon After 2 5 mL fore- run, three-1 mL fractions were collected and combined Recovery of antibody was 3 66 mg or 86% by A₂₈₀ Surfact-Amps Tween-20 (10%) was added to give 0 2% final concentration

A 1 4 mL aliquot of the thiolated antibody above (1 71 mg antibody) was immediately added to 300 μL (10 mg) of maleimidated beads prepared above plus enough 10% Tween-20 to bring final concentration of the mixture to 0 2% The tube was purged with argon and incubated overnight at room temperature in the dark To the above was added 3 4 μL of 1 M HSCH₂COOH in water After 30 mm at room temperature, 6 8 μL of ICH₂COOH (1 M in water) was added After 30 mm 3 5 mL of 0 17 M glycine, 0 1 M NaCI, 0 1 % (v/v) Tween-20, 10 mg/mL BSA, pH 9 2 was added and the beads were centrifuged (30 mm at 15,000 rpm), incubated for 3 hr in 5 mL of the same buffer, centrifuged, washed by centπfugation with three-5 mL portions of Buffer C, resuspended in 5 mL of Buffer C and stored under refrigeration The size of the beads, determined in Buffer C, was 301+/-56 nm Binding capacity was determined with ¹²⁵l- digoxm and was equivalent to 377 antibody molecules per bead

Silicon tetra-t-butyl phthalocyanine was prepared as follows

Sodium metal, freshly cut (5 0 g, 208 mmol), was added to 300mL of anhydrous ether in a two-liter, 3-necked flask equipped with a magnetic stirrer, reflux condenser, a drying tube and a gas bubbler After the sodium was completely dissolved, 4-t-butyl-1 ,2- dicyanobenzene (38 64 g, 210 mmol, from TCI Chemicals, Portland OR) was added using a funnel The mixture became clear and the temperature increased to about 50°C At this point a continuous stream of anhydrous ammonia gas was introduced through the glass bubbler into the reaction mixture for 1 hr The reaction mixture was then heated under reflux for 4 hr while the stream of ammonia gas continued During the course of the reaction, as solid started to precipitate The resulting suspension was evaporated to dryness (house vacuum) and the residue was suspended in water (400mL) and filtered The solid was dried (60°C, house vacuum, P₂0₅) The yield of the product (1 ,3- dnminoisoindoline, 42 2 g) was almost quantitative This material was used for the next step without further purification To a one-liter, three-necked flask equipped with a condenser and a drying tube was added the above product (18 g, 89 mmol) and quinoline (200 mL, Aldπch Chemical Company, St Louis MO) Silicon tetrachloπde (11 mL, 95 mmol, Aldπch Chemical Company) was added with a syringe to the stirred solution over a period of 10 minutes After the addition was completed, the reaction mixture was heated to 180-185°C in an oil bath for 1 hr The reaction was allowed to cool to room temperature and concentrated HCI was carefully added to acidify the reaction mixture (pH 5-6) The dark brown reaction mixture was cooled and filtered The solid was washed with 100 mL of water and dried (house vacuum, 60°C, P₂0₅) The solid material was placed in a 1 -liter, round bottom flask and concentrated sulfunc acid (500 mL) was added with stirring The mixture was stirred for 4 hr at 60°C and was then carefully diluted with crushed ice (2000 g) The resulting mixture was filtered and the solid wad washed with 100 mL of water and dried The dark blue solid was transferred to a 1 -liter, round bottom flask, concentrated ammonia (500 mL) was added, and the mixture was heated and stirred under reflux for 2 hr , was cooled to room temperature and was filtered The solid was washed with 50 mL of water and dried under vacuum (house vacuum, 60°C, P₂0₅) to give 12g of product silicon tetra-t-butyl phthalocyanine as a dark blue solid 3-pιcohne (12 g, from Aldπch Chemical Company), tπ-n-butyl amine (anhydrous, 40mL) and tπ-n-hexyl chlorosilane (11 5 g) were added to 12 g of the above product in a one-liter, three-necked flask, equipped with a magnetic stirrer and a reflux condenser The mixture was heated under reflux for 1 5 hr and then cooled to room temperature The picohne was distilled off under high vacuum (oil pump at about 1 mm Hg) to dryness The residue was dissolved in CH₂CI₂ and purified using a silica gel column (hexane) to give 10g of pure product dι-(tπ-n-hexylsιlyl)-sιlιcon tetra-t-butyl phthalocyanine as a dark blue solid (MS [M-H]⁺ 1364 2, absorption spectra methanol 674nm (ε 180,000) toluene 678nm,

¹H NMR (250 MHz, CDCI₃) δ -2 4(m,12H), -1 3(m, 12H), 0 2-0 9 (m, 54H), 1 8(s, 36H), 8 3(d, 4H) and 9 6 (m, 8H) was consistent with the above expected product

Sens-SAv - Sensitizer beads coupled to Streptavidin (2300 SAv/bead)

The sensitizer beads were prepared placing 600 mL of carboxylate modified beads (Seradyn) in a three-necked, round-bottom flask equipped with a mechanical stirrer, a glass stopper with a thermometer attached to it in one neck, and a funnel in the opposite neck The flask had been immersed in an oil bath maintained at 94+ /-1°C The beads were added to the flask through the funnel in the neck and the bead container was rinsed with 830 mL of ethoxyethanol, 1700 mL of ethylene glycol and 60 mL of 0 1 N NaOH and the rinse was added to the flask through the funnel The funnel was replaced with a 24- 40 rubber septum The beads were stirred at 765 rpm at a temperature of 94+ /-1°C for 40 mm Silicon tetra-t-butyl phthalocyanine (10 0 g) was dissolved in 300 mL of benzyl alcohol at 60+/-5°C and 85 mL was added to the above round bottom flask through the septum by means of a syringe heated to 120+/-10°C at a rate of 3 mL per mm The remaining 85 mL of the phthalocyanine solution was then added as described above The syringe and flask originally containing the phthalocyanine was rinsed with 40 mL of benzyl alcohol and transferred to round-bottom flask After 15 mm 900 mL of deionized water and 75 mL of 0 1 N NaOH was added dropwise over 40 mm The temperature of the oil bath was allowed to drop slowly to 40+/-10°C and stirring was then discontinued The beads were then filtered through a 43 micron polyester filter and subjected to a Microgon tangential flow filtration apparatus (Microgon Inc , Laguna Hills, CA) using ethanol water, 100 0 to 10 90, and then filtered through a 43 micron polyester filter

Sulfo-SMCC (11 55 mg) was dissolved in 0 5 mL distilled water Slowly, during 10 sec, the above solution was added to 5 mL of stirring ammodextran (Molecular Probes, Eugene, Oregon) solution (12 5 mg/mL in 50mM MOPS, pH 7 2) The mixture was incubated for 1 hr at room temperature

To the stirring solution above was added 5 mL of 20 mg/mL (100 mg) of the sensitizer beads prepared above in distilled water Then, 1 mL of 200 mg/mL NHS (prepared fresh in 50 mM MES, pH adjusted to 6 0 with 6 N NaOH) 200 mg EDAC was dissolved in 1mL distilled water and this solution was added slowly with stirring to the sensitizer beads The pH was adjusted to 6 0 by addition of 450μL of 1 N HCI and the mixture was incubated overnight in the dark A solution of 100mg of succmic anhydride in 0 5mL of DMSO was added to the sensitizer beads and the mixture was incubated for 30 m at room temperature in the dark To this mixture was added 0 13mL 10% Tween-20 bringing the final concentration of Tween-20 to 0 1 % The beads were centrifuged for 45 m at 15,000 rpm as above The supernatant was discarded and the beads were resuspended in 10 mL of buffer (50 mM MOPS, 50 mM EDTA and 0 1 % Tween-20, pH 7 2) The mixture was sonicated to disperse the beads The beads were centrifuged for 30 mm as described above, the supernatant was discarded and the beads were resuspended This procedure was repeated for a total of three times Then, the beads were resuspended to 40 mg/mL in 2 5 mL of the above buffer, saturated with argon and Tween-20 was added to a concentration of 0 1 % The beads were stored at 4°C

Streptavidin was bound to the above beads using 25 mg streptavidin for 100 mg of beads 25 mg streptavidin (50 mg Aaston solid from Aaston, Wellesley, MA) was dissolved in 1 mL of 1 mM EDTA, pH 7 5, and 77 μL of 2 5 mg/mL SATA in ethanol was added thereto The mixture was incubated for 30 mm at room temperature A deacetylation solution was prepared containing 1M hydroxylamine-HCI, 50 mM Na₂P0₄, 25 mM EDTA, pH 7 0 0 1 mL of this deacetylation solution was added to the above solution and incubated for 1 hr at room temperature The resulting thiolated streptavidin was purified on a Pharmacia PD10 column and washed with a column buffer containing 50 mM MOPS, 50 mM EDTA, pH 7 2 The volume of the sample was brought to 2 5 mL by adding 1 5 mL of the above column buffer The sample was loaded on the column and eluted with 3 5mL of the column buffer The thiolated streptavidin was diluted to 5mL by adding 1 5mL of 50mM MOPS, 50mM EDTA, 0 1 % Tween-20, pH 7 2 5 mL of the thiolated streptavidin solution was added to 5 mL of the sensitizer beads, under argon, and mixed well The beads were topped with argon for 1 m the tube was sealed and the reaction mixture was incubated overnight at room temperature in the dark

To the above beads was added 7 5 mL of 50 mM MOPS, 50 mM EDTA, 0 1 % Tween-20, pH 7 2 to bring the beads to 1 mg/mL The remaining maleimides were capped by adding mercaptoacetic acid at a final concentration of 2 mM The mixture was incubated in the dark for 30 mm at room temperature The remaining thiols were capped by adding lodoacetic acid at a final concentration of 10 mM and the mixture was incubated at room temperature for 30 mm in the dark The beads were centrifuged for 30 mm at 15,000 rpm as above for a total of three times

EXAMPLE 1

PCR primer sequences are set forth in Table 1

Table 1

Specific primers for M tuberculosis rpoB gene mutation detection

Forward primers 1779 5'-L-GAGCGGATGACCACCCAGGACNNT-3' (SEQ ID NO 31) where L is Biotin or

Digoxigenm

1789 5'- L-CCACCCAGGACGTGGAGGCNNT-3' (SEQ IP NO 32) where L is Biotin or

Digoxigenm

Reverse primers 1963a 5'-ACCATGCTCGAGATTACGAG CCGGCACGCTCACGTGACANNA-3 ' (SEQ ID

NO 33)

1963b S'-GATCCTAGGCCTCACGTATTCCGGCACGCTCACGTGACANNA-3'

(SEQ ID NO 34)

2044a 5'-ACCATGCTCGAGATTACGAG CAGACCGATGTTGGGCCCCTNNA-3' (SEQ ID NO 35)

2044b 5'-GATCCTAGGCCTCACGTATT CAGACCGATGTTGGGCCCCTNNA-3' (SEQ ID

NO 36)

2080a 5'-ACCATGCTCGAGATTACGAG GGGTTGACCCGCGCGTACANNA-3'

(SEQ ID NO 37) 2080b 5'-GATCCTAGGCCTCACGTATT GGGTTGACCCGCGCGTACANNA-3'

(SEQ ID NO 38)

TaiM 5'-ACCATGCTCGAGATTACGAG-3' (SEQ IP NO 39)

Tail 2 5'-GATCCTAGGCCTCACGTATT-3' (SEQ IP NO 40)

N = etheno dA modification The position of hybridization of the primers to the rpoB gene sequence is indicated by the primer number The number indicates the target nucleotide complementary to the 5'-end of the primer (shown in bold) In the case of the reverse primers the position is related to the complementary sequence only not including tail 1 or tail 2

The above sequences correspond to SEQ ID NO 1 to 10, respectively, except for the modification at the 3'-end of each, wherein "NN" represents two etheno-dA moieties

The positions of the forward and reverse PCR primers are denoted in Fig 4, detailing the full sequence of the rpoB gene (GenBank Accession No 012205) (SEQ ID NO 11) The forward PCR primers are 5'-labeled with biotin or digoxigenm (Dig ) The reverse PCR primers are composed of two parts The 3'-portιon of the primers is identical 3 1

and is complementary to the target DNA (shown in bold is Fig 4) The 5'-portιon (identified as tail 1 and tail 2) of the two primers is different and is not related to the target DNA sequence These sequences (tail 1 and tail 2) are designed to form the tails of the heteroduplexes, which upon annealing result in the formation of a four-stranded DNA structure or quadramolecular complex

All forward and reverse primers were also modified at the 3'-end by the addition of two ethenodA residues to assist in the hot start procedure discussed below The 3'-termιnal residue was added for convenience of oligonucleotide synthesis

PCR amplification of the rpoB gene sequence PCR amplification of the rpoB gene sequence was carried out using a hot start procedure referred to as the wax bead-based method, in which commercially available PCR gems (AmpliWax from Perkin Elmer) were utilized The choice of primers and conditions for PCR amplification provided for specific and efficient production of PCR derived substrates for subsequent analysis The high GC content of the specific sequence of M tuberculosis rpoB gene also influences the effectiveness of amplification The amplification conditions described in the following section were selected for maximum specificity of the present detection method

PCR amplification of test target was carried out using 5'-bιotιn-labeled forward primer and two related reverse primers The reference target, wild type M tuberculosis rpoB gene, was amplified using the corresponding 5'-dιgoxιgenιn labeled forward primer and the same set of reverse primers as for the test target amplification

PCR amplification with wax bead-based hot start was carried as follows A master mixture (Mix 1) containing 10 mM Tπs-HCI pH 8 3 50 mM KCI, 1 5 mM MgCI₂, 0 2 mg/ml BSA, 200μM of each of the four dNTPs, and 250 nM of each of the primers was prepared 25 μl of Mix 1 was added to PCR tubes containing a wax gem, and the tubes were incubated at 80°C for 2 minutes to melt the wax gems The reaction tubes were then cooled to room temperature to form the wax barrier on top of the liquid reaction mixture A second reaction mixture containing 10 mM Tπs-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI₂, 0 2 mg/ml BSA and 2 5 U/25 μl of Pfu DNA polymerase was also prepared 20 μl of Mix 2 and 5 μl of test or reference target were added to each of the reaction tubes prepared as above PCR amplification was carried out in Tπo-Thermoblock™ thermocycler (Biometra Inc ) The thermocycle program was as follows 4 mm at 95°C, followed by 40 cycles of 45 sec at 95°C, and 2 mm at 70°C Mutation analysis and detection were performed as follows 1 μl of PCR amplification reaction mixture of reference target and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100 mM Tπs-HCI pH 8 3, 500 mM KCI, 15 mM MgCI₂ and 2 mg/ml BSA) The mixtures were subjected to one cycle of 2 mm at 95°C and 30 mm at 65°C in a thermocycler 50 μl of a bead mixture (2 5 μg of streptavidin-coated sensitizer beads and 1 25 μg of anti-digoxigenin-coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm Signal was read (3 cycles of 1 sec illumination and 1 sec read)

The results of the analysis of two wild type and two rifampin resistant M tuberculosis clinical isolates are shown in Table 2

Table 2

Detection of mutations in rpoB gene of M tuberculosis Genomic DNA purified from four clinical isolates was tested using two sets of specific primers

Exp# Amphcon size Signal

(base pairs) WT1 WT2 Mut1 Mut2

1 213-bp 39314 34502 386604 454362

2 295-bp 46342 44990 372134 577638 3 331-bp 47320 43540 298460 338174

4 223-bp 40270 40108 987332 937784

5 305-bp 41177 40484 896750 1086440

6 341-bp 43380 27396 1041370 949414

Experiments numbered (Exp#) 1 , 2 and 3 were carried out using primer sets composed of forward primer 1789 (biotin- or digoxm-labeled) and each of the three reverse primer sets

Experiments numbered 4, 5 and 6 were carried out using primer sets composed of forward primer 1779 (biotin- or digoxm-labeled) and each of the three reverse primer sets

The results of analysis using six sets of primer pairs indicated that the signals obtained with different amplification products provide good discrimination between mutant and wild type genotypes The higher signals for mutant 1 and 2, obtained in experiments 4, 5, and 6, reflect higher amplification efficiency as shown by standard gel analysis Analysis of rifampin resistant M tuberculosis was assessed using genomic DNA purified from two wild type M tuberculosis isolates two resistant M tuberculosis clinical isolates, and other Mycobacterium sp , M smegmatis M kansasn M bovis and M intracellularea Analysis was performed using two sets of forward and reverse primers, as indicated Under conditions leading to clear detection of the resistant M tuberculosis isolates, signals obtained from the non-tuberculosis mycobacteria were similar to background signals These results (Fig 5) demonstrate the high specificity of the optimized procedure Gel analysis of the amplification products revealed that the PCR primers used for the instant genotypic detection of M tuberculosis rpoB mutations were specific for amplification of M tuberculosis and the related M bovis isolates, but not non- tuberculosis Mycobacteria In so far as the rpoB gene is highly conserved, it is possible that positive signals will be obtained from mixed infection clinical specimens if the PCR conditions and primer sets used results in amplification of all rpoB gene sequences The high specificity of the present method eliminates the possibility of false positive signals due to mixed infections

Example 2 Direct genotypic detection of M tuberculosis rifampin resistance was carried out using cells grown in culture M tuberculosis clinical isolates grown in culture were suspended in 1X or 10X buffer (100 mM Tπs-HCI pH 8 3, 500 mM KCI, 15 mM MgCI₂ and 2 mg/ml BSA) (IHBB) and heat inactivated by boiling for 30 mm at 95°C Direct analysis was achieved following sonication (12-15 pulse on Branson Sonιfιer 450 ), heat-treatment (98°C for 15-mιn , using thermocycler) or treatment in a microwave (25 sec to 1-mιn at high power setting) The direct analysis of 10 M tuberculosis clinical isolates, obtained using cell suspensions pretreated as detailed above, are shown in Fig 6, demonstrating the feasibility of direct genotypic detection procedure The procedure is suitable for use in a clinical microbiology laboratory

Comparison of results (see Table 3) obtained by culture-based phenotypic determination of rifampin susceptibility (reference laboratory) and genotypic detection revealed a single discrepancy a clinical isolate determined as antibiotic susceptible by the culture-based test scored as resistant by the method of the present invention It is possible that the positive result obtained by the present analysis reflects the presence of a silent mutation, as was previously shown (Mutations in the rpoB gene of M tuberculosis that interfere with PCR-Singie strand conformation polymorphism analysis for rifampin susceptibility testing (Kim B -J et al , J Clinical Microbiology (1997) 35 492) The high specificity of the instant genotypic detection method assures the detection of all resistant isolates, a feature most important for the control of mycobacterial infection Table 3

Results obtained with heat killed M. tuberculosis isolates: heat killed cells were disrupted by sonication

Phenotype

Isolate RLU-1 RLU-2*^* g< snotype (Ref.

Lab)

1 16824 8840 wt susceptible

2 755440 1434180 mutant resistant

3 17040 9724 wt susceptible 4 626394 1495320 mutant resistant

5 11346 10980 wt susceptible 6 1065130 1363690 mutant resistant

7 11 12210 1227160 mutant resistant

8 1237660 1421840 mutant susceptible

9 1024550 1047470 mutant resistant

10 29212 10208 wt susceptible

RLU-1^* = relative luminescence unit for M. tuberculosis cells heat killed in Buffer A RLU-2*^* = relative luminescence unit for M. tuberculosis cells heat killed in Buffer B

Example 3

Detection of genotype of M. tuberculosis associated with rifampin resistance As shown in previous examples, the branch migration method was effective in the detection of rpoB gene alteration. Signals were indicative of sequence alteration of test sequence relative to reference sequence. Low signals indicated identity of the test sequence to reference sequence. However, since the signal was related also to the amount of test target relative to the reference target, low input test target may also result in low signal, which may lead to false determination of mutant genotype as a wild type genotype.

The following experiments demonstrate a method for detection of genotype associated with rifampin resistance using a normalization of the signals relative to input test target. The normalization method is based on formation of stable four stranded DNA structures when test target amplification products are mixed with similarly produced products of amplification of non-relevant reference sequence. In so far as the two sequences are not related, signals are produced from all test samples, regardless of the specific genotype. The ratio of signals produced with relevant sequence to those produced with non-relevant sequence are the normalized signals and represent test genotype regardless of input target sequence.

As shown below, 46 samples of M. tuberculosis genomic DNA purified from clinical isolates were tested in a "blinded" fashion for rpoB genotype. The signals were produced as described in the detailed protocols. The test DNA amplification products were tested against relevant wild type target genomic DNA and non-relevant reference sequence, in this case amplification products of the human cystic fibrosis gene. Amplification of the non-relevant reference sequence was carried out with reverse primers designed for branch migration analysis and composed of a 3' target specific portion and 5' tails, which were the same as those used for the M. tuberculosis rpoB analysis. This feature is important for the ability of forming four stranded structures between the test and non- relevant amplification products.

The results of the blinded study are summarized in Table 4. The ratio of signal obtained with relevant reference to signal obtained with non-relevant reference clearly differentiates mutant and wild type isolates. The mutant and wild type genotype determinations by this method are in full agreement with phenotypic determination of the clinical isolates as rifampin resistant or sensitive. Protocol for M. tuberculosis rpoB gene mutation detection

PCR amplification of the rpoB gene sequence was carried out using one of two hot start procedures: one such procedure was the wax bead-based method using commercially available PCR gems (AmpliWax from Perkin Elmer), the other such procedure involved the use of an anti-etheno A monoclonal antibody, which binds to the primers, which comprise etheno-dA moieties at their respective 3-'ends, until the temperature of the reaction medium is raised whereupon the antibody dissociates from the primers and is denatured. The choice of primers and conditions for PCR amplification are chosen for specific and efficient production of PCR derived substrates for subsequent branch migration analysis in accordance with the present invention. The high GC content of the specific sequence of M. tuberculosis rpoB gene also influenced the effectiveness of amplification. The amplification conditions described in the following were selected for maximum specificity of the present mutation detection method.

PCR amplification of test target was carried out using 5'-biotin labeled forward primer and two related reverse primers. The reference target, wild type, was amplified using the corresponding 5'-dig labeled forward primer and the same set of reverse primers as for the test target amplification. PCR amplification with wax bead based hot start was carried out as follows A master mixture (Mix 1) containing 10 mM Tπs-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI₂, 0 2 mg/ml BSA, 0 5 μM of each of the four dNTPs, and 0 5 μM of each of the primers, was prepared 25 μl of Mix 1 was added to PCR tubes containing a Wax gem, and the tubes were incubated at 80°C, for 2 minutes, to melt the wax gems The reaction tubes were then cooled to room temperature, to form the wax barrier on top of the liquid reaction mixture A second reaction mixture (Mix 2) containing 10 mM Tπs-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 2 mg/ml BSA and 2 5 U/25 μl of Pfu DNA polymerase, was also prepared 20 μl of Mix 2 and 5 μl of test or reference target were added to each of the reaction tubes prepared as above PCR amplification was carried out in Tπo-Thermoblock thermocycler (Biometra Inc , Tampa, Fl) The thermocycle program was as follows 4 mm at 95°C followed by 40 cycles of 45 sec at 95 °C, and 2 mm at 70°C

PCR amplification using the antibody-based hot start procedure was carried out as follows a master reaction mixture containing 10 mM Tπs-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 0 25 mM of each of four dNTPs, 0 2 mg/ml BSA, 0 5 μM anti etheno A monoclonal antibody (from Inst fur Zelibiologie, Dr Petra Lorenz), and 1 25 U/25 μl Pfu DNA polymerase, was prepared 2 5 μl of test or reference target was added to 22 5 μl of the master reaction mix, in PCR tubes PCR amplification was carried out using conditions similar to the above PCR amplification of non-relevant target was carried out using the antibody-based hot start procedure as described above All of the reagents were the same as stated above except the target was cystic fibrosis exonl 1 (wild type) and the primers were a mixture of 5'-bιotιn and 5'-dιg labeled forward primers and a mixture of reverse primers modified with the same taιl-1 and taιl-2 and 3'-end etheno-dA modified nucleotides as for M tuberculosis as described above The primers are identified in the CFTR Gene, Exon 11 Sequence below

Analysis and detection were performed as follows (1) For genotype determination

1 μl of PCR amplification reaction mixture of reference target and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100mM Tπs-HCI pH8 3, 500mM KCI, 15mM MgCI2 and 2mg/ml BSA) The mixtures were subject to one cycle of 2 mm at 95°C and 30 m at 65°C, in a thermocycler 50 μl of a bead mixture (2 5μg of streptavidm-coated sensitizer beads and 1 25 μg of anti-Dig-coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm and signal was read (3 cycles of 1 sec illumination and 1 sec read) (2) For DNA Confirmation

1 μl of PCR amplification reaction mixture of PCR amplified non-relevant target of wild type CF at 1/10 dilution and 1 μl of test PCR amplification reaction mixture were added to PCR tubes containing 4 μl buffer (100mM Tπs-HCI pH8 3, 500mM KCI, 15mM MgCI₂ and 2mg/ml BSA) The mixtures were subject to one cycle of 2 m at 95°C and 30 mm at 65°C, in a thermocycler 50 μl of a bead mixture (2 5 μg of streptavidm-coated sensitizer beads and 1 25 μg of anti-Dig-coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm and signal was read (3 cycles of 1 sec illumination and 1 sec read)

Blind studies of M tuberculosis rpoB gene mutation detection

46 samples of M tuberculosis genomic DNA were tested for the genotype indicative of rifampin resistance using an assay in accordance with the present invention followed by detection with anti-dig Ab coated acceptor chemiluminescent beads and streptavidin coated sensitizer beads The results are summarized in Table 4

Table 4

CFTR Gene, Exon 11 Seguence

1 atatacccat aaatatacac atattttaat ttttggtatt ttataattat tatatgggta tttatatgtg tataaaatta aaaaccataa aatattaata

51 tatttaatga tcattcatga cattttaaaa attacaggaa aaatttacat ataaattact agtaagtact gtaaaatttt taatgtcctt tttaaatgta

101 ctaaaatttc agcaatgttg tttttgacca actaaataaa ttgcatttga gattttaaag tcgttacaac aaaaactggt tgatttattt aacgtaaact

151 aataatggag atgcaatgtt caaaatttca actgtggtta aagcaatagt ttattacctc tacgttacaa gttttaaagt tgacaccaat ttcgttatca f2-B Biotin-5 '-tag aaggaagatg tgcctttca-3 ' (SEQ ID Nθ:14) f2-D Digoxin-5 '-tag aaggaagatg tgcctttca-3^{^} (SEQ ID Nθ:14)

201 gtgatatatg attacattaq aaqqaaqatq tqcctttcaa attcagattg cactatatac taatgtaatc ttccttctac acggaaagtt taagtctaac

251 agcatactaa aagtgactct ctaattttct atttttggta ataggacatc tcgtatgatt ttcactgaga gattaaaaga taaaaaccat tatcctgtag

542

301 tccaagtttg cagagaaaga caatatagtt cttGGAqaaq gtggaatcac aggttcaaac gtctctttct gttatatcaa gaacctcttc caccttagtg

551 553 560

351 actgagtgga GGTcaaCGAg caagaatttc tttaqcaAGG tgaataacta tgactcacct ccagttgctc gttcttaaag aaatcgttcc acttattgat 401 attattggtc tagcaagcat ttgctgtaaa tgtcattcat gtaaaaaaat taataaccag atcqttcqta aac acattt acaqtaa ta cattttttta rltl 3'-cgttcgta aacgacattt acag

-ttatgcactccggatcctag-5 ' (SEQ ID NO: 15) rlt2 3'-cgttcgta aacgacattt acag -gagcattagagctcgtacca-5 ' (SEQ ID NO: 16)

451 tacagacatt tctctattgc tttatattct gtttctggaa ttgaaaaaat atgtctgtaa agagataacg aaatataaga caaagacctt aactttttta 501 cctggggttt tatggctagt gggttaagaa tcacatttaa gaactataaa ggaccccaaa ataccgatca cccaattctt agtgtaaatt cttgatattt

551 taatggtata gtatccagat ttggtagaga ttatggttac tcagaatctg attaccatat cataggtcta aaccatctct aataccaatg agtcttagac

601 tgcccgtatc ttgg 3' 614 (SEQ ID NO: 12) acgggcatag aacc 5' (SEQ ID NO: 13)

The f2/rl primer set flanks 173 bases of the CFTR Exon 11 sequence, resulting in an amplicon which includes 217 bases from Exon 11 and 20 bases from the reverse primer tails, for a total of 237 bp. Example 4

Detection of genotype of M. tuberculosis associated with pyrazinamide resistance

Genomic DNA:

Eight (8) genomic DNA preparations of M. tuberculosis pyrazinamide resistant isolates and 29 genomic DNA preparations of wild type M. tuberculosis isolates (from the rifampin sensitive group of isolates previously used) were obtained from Dr. Peter Small at Stanford Medical Center and used in this study. Genomic DNA preparation of other

Mycobacteria strains were also obtained from Stanford University (P. Small).

Target seguences: A) Sequence of M. tuberculosis pncA gene (561 -bp) (U59967)

1 atgcgggcgt tgatcatcgt cgacgtgcag aacgacttct gcgagggtgg ctcgctggcg

61 gtaaccggtg gcgccgcgct ggcccgcgcc atcagcgact acctggccga agcggcggac 121 taccatcacg tcgtggcaac caaggacttc cacatcgacc cgggtgacca cttctccggc

181 acaccggact attcctcgtc gtggccaccg cattgcgtca gcggtactcc cggcgcggac

241 ttccatccca gtctggacac gtcggcaatc gaggcggtgt tctacaaggg tgcctacacc

301 ggagcgtaca gcggcttcga aggagtcgac gagaacggca cgccactgct gaattggctg

361 cggcaacgcg gcgtcgatga ggtcgafgtg gtcggfattg ccaccgatca ttgtgtgcgc 421 cagacggccg aggacgcggt acgcaatggc ttggccacca gggtgctggt ggacctgaca

481 gcgggtgtgt cggccgatac caccgtcgcc gcgctggagg agatgcgcac cgccagcgtc

541 gagttggttt gcagctcctg a (SEQ ID NO:20) 1. The nucleotides denoted in bold letters represent sites in the pncA gene sequence of known mutations, which result in PZA resistance.

2. Only the coding sequence of the pncA gene is shown. The primers of the invention hybridize to the target at sites flanking the coding sequence. 61

B) Human Cystic Fibrosis genomic DNA exon 11 (see above)

Oligonucleotide Seguences (PCR primers 5' to 3') M tuberculosis FP/(Bιo/Dιg)39ue 5'- Bιo(Dιg)-tggtgccgcgtcggtaggnnt-3' (SEQ ID NO 25)

RP/541t1e 5'-accatgctcgacattacgagtcaggagctgcaaaccaactcnna-3' (SEQ ID

NO 26) RP/541t2e 5'-gatcctaggcctcacgtatttcaggagctgcaaaccaactcnna-3'

(SEQ ID NO 27) The above sequences correspond to SEQ ID NO 21 , SEQ ID NO 22 and

SEQ ID NO 23, respectively, except for the modification at the 3'-end, wherein "nn" represents two etheno-dA moieties

Human CFTR gene exon 11 f2-(Bιo/Dιg) Dιg-5'-tagaaggaagatgtgcctttcannt-3' (SEQ ID NO 28) r111 5'-accatgctcgagattacgaggacatttacagcaaatgcttgcnna-3'

(SEQ ID NO 29) r1t2 5'-gatcctaggcctcacgtattgacatttacagcaaatgcttgcnna-3'

(SEQ ID NO 30) The above sequences correspond to SEQ ID NO 14 SEQ ID NO 15 and

SEQ ID NO 16, respectively except for the modification at the 3'-end, wherein "nn" represents two etheno-dA moieties

The underlined portions of the above sequences represent the following oligonucleotide tails taιl-1 5'-accatgctcgagattacgag-3' (SEQ ID NO 9) taιl-2 5'-gatcctaggcctcacgtatt-3' (SEQ ID NO 10)

Methods

PCR amplification PCR amplification with wax bead based hot start was carried out as follows A master mixture (Mix 1) containing 10 mM Tπs-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 0 2mg/ml BSA, 200 μM of each of the four dNTPs, and 250 nM of each of the primer was prepared 25 μl of Mix 1 was added to PCR tubes containing a Wax gem, and the tubes were incubated at 80°C for 2 minutes to melt the wax gems The reaction tubes were then cooled to room temperature to form the wax barrier on top of the liquid reaction mixture. A second reaction mixture (Mix 2) containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCI₂, 0.2 mg/ml BSA and 2.5 U/25 μl of Pfu DNA polymerase was also prepared. 20 μl of Mix 2 and 5 μl of test DNA or reference DNA (10⁴ molecules) were added to each of the reaction tubes prepared as above. PCR amplification was carried out in the T3 thermocycler (Biometra Inc.). The following thermocycling protocol was used: The reaction mixture was first heated to 95°C for 4 min. for denaturation of the genomic DNA, followed by 39 cycles of 30 sec at 95°C, 1 min. at 64°C and 1 min. at 72°C, followed by 5 min. at 72°C for final extension. The amplification product generated by the disclosed primers is 620 bp long.

PCR amplification of non-relevant target was carried out the same way as above. All the reagents are the same as stated above except the target is cystic fibrosis exonl 1 genomic DNA (wild type) and the primers are f2/5'-dig labeled forward primer and a mixture of reverse primers modified with the same tail-1 and tail-2 (r1t1 &r1t2) as for M. tuberculosis.

Mutation analysis and detection were performed as follows: 1 μl of post-PCR amplification reaction mixture of reference target and 1 μl of post-PCR amplification reaction mixture of test target were added to PCR tubes containing 4 μl of 10x buffer (100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI2 and 2 mg/ml BSA). The mixtures were incubated for 2 min at 95°C followed by 30 min at 65°C, in a thermocycler. 50 μl of bead mixture that contained 2.5 μg of streptavidin-coated sensitizer beads and 1.25 μg of anti- dig-coated acceptor beads were added to each tube and the tubes were incubated at 37°C for 30 min. Signal was read as previously described (3 cycles of 1 sec. illumination and 1 sec. read). Normalization of the signal reading was carried out as follows: 1 μl of post-PCR amplification reaction mixture of non-relevant target (wild type Cystic Fibrosis genomic DNA, 1/10 dilution) and 1 μl of test post PCR amplification reaction mixture of test target were added to PCR tubes containing 4 μl of 10x buffer (100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI₂ and 2 mg/ml BSA). The mixtures were incubated for 2 min at 95°C followed by 30 min at 65°C, in a thermocycler. 50 μl of the bead mixture, which contained 2.5 μg of streptavidin-coated sensitizer beads and 1.25 μg of anti-dig-coated acceptor beads, were added to each tube and the tubes were incubated at 37°C for 30 min. Signal was read as above. The ratios of signals obtained from the mixtures of test post-PCR amplification reaction products and reference (wt M tuberculosis) post-PCR amplification products (S1) to the signals, similarly obtained from mixtures of the test post-PCR amplification products and the non-relevant reference post amplification products (S2), represent the normalized signals PCR products were also detected by gel electrophoresis 5 μl reaction mixture and 5 μl dye solution were mixed and subjected to electrophoresis using native 4-20% gradient polyacrylamide gel in Tπs-glycme native gel buffer followed by staining with ethidium bromide

Results

Mutations leading to PZA resistance in M tuberculosis were shown to occur throughout the pncA gene Primers which afford PCR amplification of the entire gene to generate a product suitable for gene alteration analysis were designed The sequence of the primers used is given above As for other analysis, the primers used included two forward primers having the same sequence and labeled at the 5' end with either biotin or digoxigenm, and two reverse primers each having a 3' portion that was complementary to the target and was the same for both reverse primers, and 5' tail sequences that are not complementary to the target (test or reference) and are not complementary to each other These primers generated 620 bp long amplification products The amplification products were labeled with either biotin or digoxigenm and had the tail sequences at one end For the analysis of the mycobacterium pncA gene, the test target was amplified with biotin labeled forward primer, and the reference target with a digoxigenm labeled forward primer Amplification reaction of both test and reference targets was carried out with a mixture of the two reverse primers The selected disclosed primers afforded efficient PCR amplification, as was demonstrated by gel electrophoresis (result not shown) and efficient detection of gene alterations as shown below

The conditions for PCR amplification were optimized with respect to MgCI₂ and salt concentrations Additionally, reaction carried out with Turbo Pfu polymerase resulted in reduced non-specific signals and increased specific signals as compared to reactions carried out with cloned Pfu (Fig 7) The data shown in the following was obtained using the optimized conditions

A total of 37 M tuberculosis isolates were tested in accordance with the present invention for genotypic determination of PZA susceptibility using the disclosed primers Included were 29 phenotypically wild type strains (used in the previous demonstration of determination of rifampin resistance in M. tuberculosis) and 8 phenotypically PZA resistant isolates (all obtained from Stanford University, P. Small). The genotypic determination of PZA resistance is shown in Fig. 8. The results demonstrate perfect correlation of the phenotypic and genotypic determination, with very strong discrimination of susceptible and resistant genotypes. The normalized signals of the analysis for the detection of PZA resistant M. tuberculosis are given in Table 5.

Table 5

The specificity and reproducibility of the present method for the genotypic detection of PZA resistant M. tuberculosis were also demonstrated. Genomic DNA preparations from four MOTT isolates, including M. smegmatis, M. kansasii, M. bovis and M. intracellulurea, were tested for gene alterations relative to M. tuberculosis wild type pncA gene sequence, using the disclosed primers and method. As shown in Fig. 9, only analysis of M. bovis indicated altered gene sequence. This result is in agreement with the known natural PZA resistance of M. bovis, which is a result of known gene alteration (Angelo, et al., J. Clin. Microbiology, 1997, 35, 106-1 10). Thus, the results indicate the high specificity of the new test method for genotypic determination of PZA resistance.

High reproducibility of the present method was also demonstrated as shown in Fig. 10. The reproducibility of the detection of PZA resistance was demonstrated by repeated testing (eight individual analyses) of a wild type and mutant (PZA resistant) genomic DNA samples. Mean signal of wt = 221 ,355; SD=1528.4 & CV=6.9%; Mean signal of mut = 2,283,981 ; SD=106972 & CV=4.6%

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. A portion of the present disclosure contains material that may be subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS

1 A method for detecting drug resistance in a strain of an organism, said method comprising (a) detecting the presence of at least one mutation in a first sequence representing a predetermined region within the gene of said strain, said predetermined region having a multiplicity of mutations among strains of said organism that differ from a second sequence representing a corresponding region of the wild type strain of said organism, wherein said mutation is detected by a method comprising (i) forming a complex comprising said first and said second sequences in double stranded form, wherein each member of at least one pair of non-complementary strands within said complex has a label and

(n) detecting the association of said labels within said complex, the association thereof being related to the presence of said mutation and (b) relating the presence of said mutation to the drug resistance of said strain

2 A method according to Claim 1 wherein said organism is M tuberculosis

3 A method according to Claim 2 wherein said gene is the rpoB gene

4 A method according to Claim 2 wherein said drug resistance is rifampin resistance or pyrazinamide resistance

5 A method according to Claim 1 wherein said label is independently selected from the group consisting of ohgonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

6 A method for detecting drug resistance in a strain of M tuberculosis, said method comprising

(a) detecting the presence of at least one mutation in a first sequence representing a predetermined region within the rpoB gene of said strain wherein said mutation is detected by a method comprising (i) forming from said first sequence a tailed target partial duplex A' comprised of a duplex of said first sequence, a label and at one end of said duplex, two non-complementary ohgonucleotides, one linked to each strand,

(n) providing in combination said tailed target partial duplex A' and a tailed reference partial duplex B' from a second sequence representing a corresponding region of a wild type strain having a label as a part thereof, wherein said labels are present in non-complementary strands of said tailed target and tailed reference partial duplexes respectively, and

(in) detecting, by means of said labels, the formation of a complex between said tailed partial duplexes, the formation thereof being directly related to the presence of said mutation, and

(b) relating the presence of said mutation to the drug resistance of said strain of M tuberculosis

7 A method according to Claim 6 wherein said drug resistance is rifampin resistance

8 A method according to Claim 6 wherein said label is independently selected from the group consisting of ohgonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

9 A method for detecting drug resistance in a strain of M tuberculosis, said method comprising (a) detecting the presence of at least one mutation in a first sequence representing a predetermined region within a gene of said strain wherein said mutation is detected by a method comprising

(i) amplification of said first sequence by polymerase chain reaction, using primers P1 and P2 to produce an amphcon AA, wherein one of said primers P1 and P2 comprises a label and wherein said primer P1 is comprised of a 3'-end portion Pa that can hybridize with said first sequence and a 5'-end portion B1 that cannot hybridize with said first sequence,

(n) extending a primer P3 by chain extension along one strand of amphcon AA to produce a tailed target partial duplex A' wherein said primer P3 is comprised of said 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to said first sequence or its complement,

(in) amplification of a second sequence representing a region in wild type M tuberculosis, said region corresponding to said predetermined region, using said primer P2 and said primer P3, by polymerase chain reaction to produce amphcon BB, wherein said primer P2 comprises a label when said primer P2 in step (i) above comprises a label and said primer P3 comprises a label when said primer P1 in step (i) above comprises a label,

(iv) extending said primer P1 by chain extension along one strand of amp con BB to produce a tailed reference partial duplex B',

(v) allowing said tailed target partial duplex A' to bind to said tailed reference partial duplex B', and

(vi) detecting the binding of one of said labels to another of said labels as a result of the formation of a complex between said tailed partial duplexes, the binding thereof being directly related to the presence of said mutation, and

10 A method according to Claim 9 wherein said A1 and said A2 each have from 15 to 60 nucleotides

11 A method according to Claim 9 wherein said label is independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

12 A method according to Claim 9 wherein said gene is selected from the group consisting of the rpoB gene and the pncA gene

13 A method for detecting rifampin resistance in a strain of M tuberculosis, said method comprising

(a) subjecting to conditions for polymerase chain reaction a medium comprising (i) a first sequence representing a predetermined region within the rpoB gene of said strain, (n) a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO 1)and 5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO 2), and

(iii) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of said P1 and P3, wherein said common nucleotide sequence is selected from the group consisting of 5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO 17), 5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO 18) and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO 19), the product of said polymerase chain reaction being a tailed target duplex A' produced from said first sequence,

(b) subjecting to conditions for polymerase chain reaction a medium comprising (i) a second sequence representing a sequence within wild type strain that corresponds to said first sequence, (n) a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO 1) and 5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO 2), wherein two different labels are employed for each labeled primer P2 and (iii) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of said P1 and P3, wherein said common nucleotide sequence is selected from the group consisting of 5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO 17), 5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO 18) and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO 19), the product of said polymerase chain reaction being a tailed reference partial duplex B' produced from said second sequence,

(c) allowing said tailed target partial duplex A' to bind to said tailed reference partial duplex B',

(d) detecting the binding of one of said labels to another of said labels as a result of the formation of a complex between said tailed partial duplexes, the binding thereof being directly related to the presence of said mutation, and

(e) relating the presence of said mutation to the rifampin resistance of said strain of M tuberculosis

14 A method according to Claim 13 wherein said different oligonucleotide tails are 5'-ACCATGCTCGAGATTACGGAG-3'(SEQ ID NO 9) and 5'- GATCCTAGGCCTCACGTATT-3' (SEQ ID NO 10)

15 A method according to Claim 13 wherein said label is independently selected from the group consisting of o gonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

16 A method according to Claim 13 wherein said labels are biotin and digoxigenm

17 A method according to Claim 13 wherein said method is repeated except that said second sequence is a non-relevant reference sequence in a predetermined amount and said 5'-labeled primers and said 5'-taιled primers comprise said oligonucleotide tails and priming sequences for said non-relevant reference sequence and a ratio is obtained of the signal obtained from the labels employed in said method of Claim 13 to the signal obtained from the labels in said repeated method

18 A method for detecting pyrazinamide resistance in a strain of M tuberculosis, said method comprising

(a) subjecting to conditions for polymerase chain reaction a medium comprising (i) a first sequence representing a predetermined region within the pncA gene of said strain, (n) a 5'-labeled primer P2, which is 5'- L-tgg tgc cgc gtc ggt agg-3' (SEQ ID NO 21), wherein two different labels are employed for each labeled primer P2 and (in) 5'-taιled primers P1 and P3 5'-acc atg etc gac att acg agt caggagctgcaaaccaactc-3' (SEQ ID NO 22) 5'-gat cct agg cct cac gta ttt cag gagctgcaaaccaactc-3' (SEQ ID NO 23) comprising a common nucleotide sequence, i e , a sequence that is the same in both P1 and P3, and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is 5'-t caggagctgcaaaccaactc-3' (SEQ ID NO 24), the product of said polymerase chain reaction being a tailed target duplex A' produced from said first sequence, (b) subjecting to conditions for polymerase chain reaction a medium comprising (i) a second sequence representing a sequence within wild type strain that corresponds to said first sequence, (n) a 5'-labeled primer P2, which is 5'- L-tgg tgc cgc gtc ggt agg-3' (SEQ ID NO 21), wherein two different labels are employed for each labeled primer P2 and (in) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of said P1 and P3, wherein said common nucleotide sequence is

5'-t caggagctgcaaaccaactcnna-3' (SEQ ID NO 24), the product of said polymerase chain reaction being a tailed reference partial duplex B' produced from said second sequence,

(e) relating the presence of said mutation to the pyrazinamide resistance of said strain of M tuberculosis

19 A method according to Claim 18 wherein said different oligonucleotide tails are 5'-acc atg etc gag att acg ag-3' (SEQ ID NO 9) and

5'-gat cct agg cct cac gta tt-3' (SEQ ID NO 10)

20 A method according to Claim 18 wherein said label is independently selected from the group consisting of o gonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

21 A method according to Claim 18 wherein said labels are biotin and digoxigenm

22 A method according to Claim 18 wherein said method is repeated except that said second sequence is a non-relevant reference sequence in a predetermined amount and said 5'-labeled primers and said 5'-taιled primers comprise said oligonucleotide tails and priming sequences for said non-relevant reference sequence and a ratio is obtained of the signal obtained from the labels employed in said method of Claim 18 to the signal obtained from the labels in said repeated method

23 A kit for carrying out a method for detecting rifampin resistance in a first sequence representing a strain of M tuberculosis, said kit comprising

(a) a second sequence representing a sequence within wild type strain that corresponds to said first sequence,

(b) a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO 1) and 5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO 2), wherein two different labels are employed for each labeled primer P2, and

(c) 5'-taιled primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of said P1 and P3, wherein said common nucleotide sequence is selected from the group consisting of 5'-CCGGCACGCTCACGTGACA-3' (SEQ ID NO 17),

5'-CAGACCGATGTTGGGCCCCT-3' (SEQ ID NO 18) and 5'-GGGTTGACCCGCGCGTACA-3' (SEQ ID NO 19)

24 A kit according to Claim 23 wherein said different oligonucleotide tails are 5'-ACCATGCTCGAGATTACGGAG-3' (SEQ ID NO 9) and

5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO 10)

25 A kit according to Claim 23 wherein said label is independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

26 A kit according to Claim 23 wherein said labels are biotin and digoxigenm

27 A kit according to Claim 23 further comprising reagents for carrying out the polymerase chain reaction

28 A kit according to Claim 23 further comprising a non-relevant reference sequence in a predetermined amount and 5'-labeled primers and 5'-taιled primers comprising said oligonucleotide tails and priming sequences for said non-relevant reference sequence

29 A kit for carrying out a method for detecting pyrazinamide resistance in a first sequence representing a strain of M tuberculosis, said kit comprising

(b) a 5'-labeled primer P2, which is 5'- L-tgg tgc cgc gtc ggt agg-3', wherein two different labels are employed for each labeled primer P2, and

(c) 5'-taιled primers P1 and P3

5'-acc atg etc gac att acg agt caggagctgcaaaccaactc-3' (SEQ ID NO 22) 5'-gat cct agg cct cac gta ttt cag gagctgcaaaccaactc-3' (SEQ ID NO 23) comprising a common nucleotide sequence, i e , a sequence that is the same in both P1 and P3, and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is 5'-t caggagctgcaaaccaactc-3' (SEQ ID NO 24)

30 A kit according to Claim 29 wherein said different oligonucleotide tails are 5'-acc atg etc gag att acg ag-3' (SEQ ID NO 9) and

5'-gat cct agg cct cac gta tt-3' (SEQ ID NO 10)

31 A kit according to Claim 29 wherein said label is independently selected from the group consisting of ohgonucleotides, enzymes, dyes, fluorescent molecules, chemilummescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces

32 A kit according to Claim 29 wherein said labels are biotin and digoxigenm

33 A kit according to Claim 29 further comprising reagents for carrying out the polymerase chain reaction

34 A kit according to Claim 29 further comprising a non-relevant reference sequence in a predetermined amount and 5'-labeled primers and 5'-taιled primers comprising said oligonucleotide tails and priming sequences for said non-relevant reference sequence

35 A method for detecting the presence of a difference between two related nucleic acid sequences in an organism, said method comprising

(a) forming amplification products from both of said nucleic acid sequences using a medium comprising disrupted cells from said organism,

(b) examining said amplification products in said medium for the presence of a stable four stranded complex resulting from strand exchange, and (c) determining the presence of said difference in said nucleic acid sequences based on the presence of said stable four stranded complex in said medium

36 A method according to claim 35 wherein said disrupted cells are obtained by mechanical or chemical treatment

37 A method according to claim 35 wherein said disrupted cells are obtained form cells by heat treatment, sonication, microwave treatment, or treatment with a lysing agent

38 A method according to claim 35 wherein said complex comprises both of said nucleic acid sequences in double stranded form and said complex further comprises at least one pair of non-complementary strands and each of said non-complementary strands within said complex has a label, and said method comprises

(a) subjecting said complex to conditions wherein, if a difference between said two related nucleic acid sequences is present, strand exchange in said complex ceases and wherein, if no difference between said two related nucleic acid sequences is present, strand exchange in said complex continues until complete strand exchange occurs, and

(b) detecting a first signal from the association of said labels as part of said complex, the association thereof being related to the presence of said difference

39 A method according to claim 35 wherein said difference is a mutation

40 A method according to claim 35 wherein said nucleic acid sequences are alleles

41. A kit for detecting the presence of a difference between two related nucleic acid sequences in an organism, said kit comprising in packaged combination:

(a) two oligonucleotide primers each comprising a sequence that is hybridizable to said nucleic acid sequences in a region adjacent the site of potential difference and each comprising a label wherein said labels are different, and

(b) a lysing agent.

42. A kit according to claim 41 further comprising two oligonucleotide primers each of which has a 3'-end that hybridizes to said nucleic acid sequences and each comprises a 5'-end portion that does not bind to said nucleic acid sequences wherein said 5'-end portions are different.

43. A kit according to claim 41 wherein said region flanks said site of potential difference.