WO2006022459A1

WO2006022459A1 - Primer and probe for detection of sars coronavirus, kit comprising the primer and/or the probe, and detection method thereof

Info

Publication number: WO2006022459A1
Application number: PCT/KR2004/002119
Authority: WO
Inventors: Hae-Joon Park; Young-Ju Ha; Mee-Hyein Kim
Original assignee: Mogam Biotechnology Institute
Priority date: 2004-08-23
Filing date: 2004-08-23
Publication date: 2006-03-02
Also published as: CN101006186A

Abstract

The present application relates to primer and/or probe for detection of mutated Coronavirus which is pathogen of severe acute respiratory syndrome (SARS), kit comprising the primer and/or probe, and a diagnosis method using the kit.

Description

PRIMER AND PROBE FOR DETECTION OF SARS CORONAVIRUS, KIT COMPRISING THE PRIMER AND/OR THE PROBE, AND DETECTION

METHOD THEREOF

TECHNICAL FIELD

The present invention relates to oligonucleotides for detecting SARS (Severe Acute Respiratory Syndrome) Coronavirus, a kit including a primer and/or a probe, and

a method for detecting SARS (Severe Acute Respiratory Syndrome) Coronavirus using

the concerned kit.

BACKGROUND ART

A Severe Acute Respiratory Syndrome (SARS) has broken out over Guangdong

Province in China in November 2002, spreading over the world including Hongkong,

Singapore, Vietnam, Canada, the U.S., etc. This syndrome is developed of a fever, a cough, dyspnoea, an atypical pneumonia, etc. It is supposed that an etiologic agent has

been reported to be Coronavirus variants, and mainly propagated by animals such as cats,

dogs, etc. (Marra, et al, 2003, Science, 300:1399-1404; Guan, et al, 2003, Science,

302:276-278).

It has been reported that, after a latent period of 2~7 days, 80-90 % of patients

are changed for the better, but 10-20 % of patients are deteriorated to be progressed into

the severe acute respiratory syndrome, with about 10 % of mortality. It has been now

focused on early diagnosis and quarantines since vaccines and therapeutic agents remain

to be developed. There are no persons who are recently infected with SARS in Korea. However, the possibility of its infections can't be completely ruled out because Korea is geographically in close contact with China, and personal and physical exchanges

continue to increase. In addition to the avian influenza, it is still apprehended that its variants may arise from the animals in Korea.

Diagnosis of antigen or antibody in blood by ELISA is most common in the methods for determining infection of the virus. However, detection of the virus gene

should be preferentially conducted because it is difficult to diagnose a symptom of

infection with the method within 7 days after the symptom of infection appears. A real-time PCR apparatus has been used for detecting the highly sensitive virus genes.

A RT-PCR method should be used to detect the genes because Coronavirus is a RNA

virus. The early commercially available real-time RT-PCR kits for detecting the SARS

gene were developed from the companies Roche and Artus. The kits showed a highly

sensitive detection performance, but they have problems that the used apparatus is

limited to a LightCycler from the company Roche and highly expensive. Recently, a

kit for detecting SARS Coronavirus was developed as the ABI Prism series from the company Artus. But, the kit has also problems that it is difficult to be generally used

due to the expensive cost and it takes at least 3 hours to detect the virus. Accordingly, for the purpose to solve the problems, novel kits for detecting the SARS Coronavirus

gene remain to be developed.

DISCLOSURE QF INVENTION

Accordingly, the present inventors attempted to develop a real-time RT-PCR kit

for detecting the SARS Coronavirus RNA. Primers and probes were personally designed to develop a kit whose sensitivity and specificity are maintained or improved, compared to the conventional kits. It is expected that developing the kit for detecting

the SARS Coronavirus RNA using the self-designed primer/probe will contribute much

to detecting it exactly at the early stage of its infection, and spreading the detection kit

widely in and out of the Korea will also contribute much to preventing infection of the

severe acute respiratory syndrome.

In order to accomplish the above object, the present invention provides a primer/probe for detecting SARS Coronavirus RNA with real-time RT-PCR process.

The primer of the present invention is preferably set forth in SEQ ID NO: 8 or SEQ ID NO: 9, and the probe of the present invention is preferably set forth in SEQ ID NO: 10.

In the present invention, the probe is characterized in that it is modified into FAM, TET,

or VIC at the 5' terminus and TAMRA at the 3' terminus.

Also, the present invention provides a kit for detecting SARS (Severe Acute

Respiratory Syndrome) Coronavirus, including the primer and/or the probe.

In the present invention, the kit is characterized in that it further includes DNA

polymerase or reverse transcriptase.

Also, the present invention provides a method for detecting SARS (Severe Acute Respiratory Syndrome) Coronavirus, the method including steps of mixing enzyme composition including DNA polymerase and/or reverse transcriptase with

reaction composition including the primer and/or probe; adding specimen RNA to the

mixture prepared in the previous step; and amplifying reaction solution including the

specimen RNA prepared in previous step using a RT-PCR process.

The kit of the present invention has an excellent sensitivity and specificity in detecting the SARS Coronavirus RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of

the present invention will be more fully described in the following detailed description,

taken accompanying drawings. In the drawings:

Fig. 1 is a schematic view showing a real-time RT-PCR principle;

Fig. 2 shows a gene map of SARS Coronavirus and a location of an amplicon

RNA on a genome;

Fig. 3 is an electrophoretic view showing standard positive RNA detected by 7

M urea/6 % PAGE (polyacrylamide gel electrophoresis);

Figs. 4a and 4b are diagrams showing a result of a real-time RT-PCR using a

primer set 1 ; In the figure, Fig. 4a shows a PCR graph, and Fig. 4b shows a standard

curve. The term "ntc" represents a non-template control, and STDl, STD2, STD3 and

STD4 represent that IXlO¹ copies/μl, IXlO² copies/μl IXlO³ copies/μl and IXlO⁴

copies/μl of amplicon RNA are used as a reaction template, respectively.

Fig. 5a and 5b are diagrams showing a result of a real-time RT-PCR using a primer set 2; In the figure, Fig. 5a shows a PCR graph, and Fig. 5b shows a standard curve. The term "ntc" represents a non-template control, and STDl, STD2, STD3 and

copies/μl of amplicon RNA are used as a reaction template, respectively.

Fig. 6a and 6b are diagrams showing a result of a real-time RT-PCR using a

primer set 3; In the figure, Fig. 6a shows a PCR graph, and Fig. 6b shows a standard curve. The term "ntc" represents a non-template control, and STDl₅ STD2, STD3 and

copies/μl of amplicon RNA are used as a reaction template, respectively.

Fig. 7a and 7b are diagrams showing a result of a real-time RT-PCR using a BNI primer set as a positive control; In the figure, Fig. 7a shows a PCR graph, and Fig.

7b shows a standard curve. The term "ntc" represents a non-template control, and

STDl, STD2, STD3 and STD4 represent that IXlO¹ copies/μl, IXlO² copies/μl IXlO³

copies/μl and IXlO⁴ copies/μl of amplicon RNA are used as a reaction template,

respectively.

Fig. 8 is a table showing clinical data about the positive specimen;

Fig. 9a and 9b are diagrams showing a result of a real-time RT-PCR about the

positive specimen; In the figure, Fig. 9a shows a PCR graph, and Fig. 9b shows a

standard curve. The term "ntc" represents a non-template control, and STDl, STD2,

STD3 and STD4 represent that IXlO¹ copies/μl, IXlO² copies/μl IXlO³ copies/μl and

IXlO⁴ copies/μl of amplicon RNA are used as a reaction template, respectively.

Fig. 10 is a table showing clinical results; and

Fig. 11 is a diagram showing a result of a real-time RT-PCR using a primer set

4 as a negative control. In the figure of the primer set 4, the term "ntc", STDl, STD2, STD3 and STD4 have the same concentration of the template as in Figs. 4, 5, 6 and 7.

That is to say, the term "ntc" represents a non-template control, and STDl, STD2, STD3

and STD4 represent that IXlO¹ copies/μl, IXlO² copies/μl IXlO³ copies/μl and IXlO⁴

copies/μl of amplicon RNA are used as a reaction template, respectively. It is seen

from Fig. 11 that combination of the primer and the probe is not detected in the primer set 4 at all.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. In order to produce a standard positive RNA specimen of the present invention,

amplicon RNA, recommended by BNI (Bernhard-Nocht Institute) in Germany, was

prepared (see Fig. 2). The amplicon RNA is located at a site of nucleotides

18120-18421 on the viral RNA genome, whose length correspond to 302 base pairs

(bp). The RNA was transcribed in vitro in a cloned vector and then purified from the

urea/PAGE (see Fig. 3). Concentration of the purified RNA was quantitatively

calculated using UV spectrophotometer, and standard RNA of the conventional

commercially available real-time RT-PCR kit.

Three sets of primers/probes were designed to detect the standard specimen RNA of SARS Coronavirus. And, the primers/probes and the real-time RT-PCR

recommended by BNI were used to compare their sensitivities and effectivenesses (see

Figs. 4 to 7). The term "ntc" represents a non-template control, and STDl, STD2,

IXlO⁴ copies/μl of amplicon RNA are used as a reaction template, respectively. A

threshold was set to 0.02 in various primer sets to analyze slopes, intercepts and

correlation values of the standard curves. It was meant that reliability is excellent as

the correlation value approaches -1. In the case of the primer set 1 (SEQ ID NOs: 3, 4

and 5) and the primer set 2 (SEQ ID NOs: 5, 6 and 7), the correlation values of the

standard curves were -0.99760 and -0.98930, respectively. It was assumed that both primer sets showed the ideal correlation values, but some non-specific bonds were formed between the primers, or the primer and the probe in reference to the high

background of the ntc (see Figs. 4 and 5). In the case of the primer set 3 (SEQ ID NOs: 8, 9 and 10), the correlation value is -0.99586, which shows excellent reliability,

and the background of the ntc was also formed ideally (see Fig. 6). In the case of the

BNI primer set (SEQ ID NOs: 11, 12 and 13), it was estimated that it is difficult to

detect low copies of viral RNA because the standard RNAs such as the STDl and the

ntc were hardly discerned due to their lowest concentration (see Fig. 7). Also in the

primer set 4 used as the control primer, a sense primer is 5'-CCTCTCTTGT TCTTGCTCGC-S¹ (SEQ ID NO: 14), and an antisense primer is 5'-ATAGTGAGCC

GCCACACATG-3' (SEQ ID NO: 15), which correspond to sequences 15,271^st- 15,290^th

(forward), 15,371^st -15,390^th (reverse) of the SARS-CoV, and the used probes were

identical to the probes used in the primer sets 1 and 2.

Finally, it was estimated that the primer sets 1, 2 and 3 may be used for

detection of SARS Coronavirus, and it may revealed that, especially among the primer

sets 1, 2 and 3, the primer set 3 showed the excellent reliability and sensitivity for

detection, compared to the primer sets of the other base sequences. Positive clinical specimens were obtained from a Chinese Center for Disease Control and Prevention to examine whether or not the viral RNA from real SARS Coronavirus patients may be

detected in an effective manner using the primer set 3 (see Fig. 8). The viral RNAs

isolated and purified from the feces of the patients were used to conduct the real-time RT-PCR (see Fig. 9). A R² value is shown to be 0.94096 when the threshold was set to

699. RNA copy numbers of the clinical specimens were counted by conducting the examinations in duplicate, based on the standard curve (see Fig. 10). For positive/negative detections, the RNA copy number is presented as (+) if RNA is present at an amount of more than 4 copies/ml, and presented as (-) if RNA is present at

amount of less than 4 copies/ml. Among the 15 test samples, 10 test samples were

presented as (+/+), 4 test samples were presented as (+/-), and 1 test sample was presented as (-/-). It was determined that it showed an excellent detection rate of

99.3 %, considering that detection rate of the product developed by the company DaAn in China is 66.7 %.

Hereinafter, the present invention will be described in detail with reference to

non-limiting Examples. Example 1

Preparation of a Plasmid for Expressing Amplicon RNA of SARS Coronavirus

A single-strand DNA was prepared by means of an oligomer synthesis, which

corresponds to 302 nucleotides (nts) of amplicon RNA. The DNA was PCR amplified using the primers T7AMPF (SEQ ID NO: 1) and AMPR (SEQ ID NO: 2), and then

purified using Qiaquick PCR purification kit (Cat. No. 28106) from the company Qiagen. The purified PCR product was cloned using pGEM-T easy vector (Cat. No. Al 360) from the company Promega. The finally cloned inserts were identified by means of DNA sequencing.

Example 2

In vitro Transcription and Purification of SARS Coronavirus Amplicon RNA

Megascript T7 kit (Cat. No. 1334) from the company Ambion was used in an in vitro transcription reaction (see an appended manual). Transcripts were located on a

7M urea/ 6 % polyacrylamide gel by the electrophoresis in a TBE buffer, followed by

ethidium bromide staining, and then purified. A purification process is carried out, as

follows. 0.4 ml of a RNA gel extraction buffer/1 band was treated and kept at 37 ^°C

for 2 hours. Supernatants was then taken to treat 0.1 ml of RNA gel extraction

buffer/1 lane, kept at 37 ^°C for 30 minutes, extracted with phenol (Ix volume), and

then precipitated with ethanol. Ethanol (3 x volume) was then added to a pellet present

in 50 μl of 0.2 M KOAc, precipitated with ethanol, and then the pellet was treated with

100 μl of RNase/Dnase-free water.

The composition of the gel extraction buffer (100 ml) is as follows: 3.85 g of

NH₄OAc + 214 mg of Mg (OAc)₂ + 200 μl of 0.5 M EDTA + 1 ml of 10 % SDS.

Example 3

Purification of viral RNA from Patient's Test Sample A QIAamp viral RNA mini kit (QIAGEN, 52 904) was used to isolate virus

RNA from the patient's feces.

Example 4

Real-time RT-PCR Additives of the final products and reaction conditions of the real-time RT-PCR

are as follows.

A required suitable amount of a cocktail was prepared by mixing 3 μl/ml of

1OX enzyme mixture with 17 μl/ml of a reaction mixture, and then divided at an amount of 20 μl. 10 μl of the specimen RNA was added to each aliquots of the cocktail to be a

final amount of 30 μl. In the RT-PCR reaction, the final mixture was kept at 50 "C

for 30 minutes, and at 95 ^°C for 10 minutes; and amplified by repeated 45 cycles at

95 ^°C for 15 seconds, at 65 ^°C for 1 minute. At this time, their fluorescent

absorbance was measured every cycle.

The composition of the reaction mixture (17 μl) is as follows: 0.3 μl of 25 μM

dNTP + 0.3 μl of the forward primer (SEQ ID NO: 8) of the 50 μM set 3 + 0.3 μl of the

reverse primer (SEQ ID NO: 9) of the 50 μM set 3 + 0.3 μl of the probe (SEQ ID NO:

10) of the 25 μM set 3 + 3 μl of a 1OX Taq Pol buffer + 12.8 μl of RNase/DNase free

water.

The composition of the enzyme mixture (10 μl) is as follows: 0.5 μl of Taq

DNA polymerase (5 u/μl) + 0.5 μl of AMV-RT (10 u/μl) + 0.5 μl of an RNase inhibitor

(40 u/μl) + 8.5 μl of an enzyme storage buffer.

The PCR amplification was carried out in the same manner as in the process using the primer set 3, except that the primer sets 1, 2 and 4, and BNI primer were used in Figs. 4, 5, 7 and 11, respectively, and SEQ ID NO: 5 was used as the probe in the real-time RT-PCR process. The results are shown in Fig. 4 (primer set 1), Fig. 5 (primer set 2), Fig. 6 (primer set 3), Fig. 7 (BNI primer) and Fig. 11 (primer set 4),

respectively.

INDUSTRIAL APPLICABILITY

As described above, the base sequences of the primers, designed to detect SARS

Coronavirus RNA using the real-time RT-PCR, may be useful to quickly and accurately detect whether or not the patients are infected with SARS Coronavirus from their

specimens. It may be directly applied to develop the real-time RT-PCR kit for detecting the SARS Coronavirus RNA using the TaqMan probe system. Therefore, it is expected to play an important role in preventing the SARS disease and its infection

that undergo a route of respiratory infection.

Claims

What is claimed is:

1. A primer for detecting SARS (Severe Acute Respiratory Syndrome) Coronavirus, set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,

SEQ ID NO: 8 or SEQ ID NO: 9.

2. A probe for detecting SARS (Severe Acute Respiratory Syndrome) Coronavirus, set forth in SEQ ID NO: 5 or SEQ ID NO: 10.

3. A kit for detecting SARS (Severe Acute Respiratory Syndrome)

Coronavirus comprising the oligonucleotides defined in the claim 1 or 2.

4. The kit for detecting SARS (Severe Acute Respiratory Syndrome)

Coronavirus according to the claim 3, wherein it further comprises DNA polymerase or

reverse transcriptase.

5. A method for detecting SARS (Severe Acute Respiratory Syndrome)

Coronavirus, the method comprising: a) mixing enzyme mixture comprising DNA polymerase and/or reverse

transcriptase with reaction mixture comprising the oligonucleotides defined in the claim l or 2;

b) adding specimen RNA to the mixture prepared in the step a); and c) amplifying reaction solution comprising the specimen RNA prepared in the step b) using a RT-PCR process.