KR100625325B1 - Probe Composition and Primer Mixture for Detection of Corona Virus and Method for Detecting Corona Virus Using the Same - Google Patents

Abstract

The present invention relates to a probe composition and a primer for detecting a coronavirus and a method for detecting a coronavirus using the same, and more specifically, to a SARS virus using a leader sequence of a SARS virus and a base sequence encoding an N protein as targets for detection. It relates to a probe composition and a primer derived from a nucleotide sequence encoding a leader sequence or N protein of SARS virus designed to effectively detect the SARS virus and a method for effectively detecting SARS virus using the probe composition or the primer.

When a combination of a coronavirus detection primer and a high heel primer according to the present invention is used, only a leader sequence of a SARS virus or a nucleotide sequence encoding an N protein can be specifically amplified to effectively detect a coronavirus including a SARS virus. It is possible.

SARS, coronavirus, reverse transcription, specific amplification, leader sequence, N protein

Description

Probe Composition and Primer Mixture for Detection of Corona Virus and Method for Detecting Corona Virus Using the Same}             

1 is a schematic diagram showing the mechanism by which coronaviruses replicate in infected cells.

Figure 2 shows a specific amplification process of the SARS virus leader sequence using a high-heel primer (high-heel) primer according to the first embodiment of the present invention.

Figure 3 shows a specific amplification process of the nucleotide sequence encoding the SARS virus N protein using a high heel primer according to a second embodiment of the present invention.

Figure 4 shows the amplification of the sample RNA by electrophoresis after performing RT-PCR using the primer mixture according to the first embodiment of the present invention. Lane M is the size marker, lane 1 is the sample RNA itself, lane 2 is the PCR amplification of the sample RNA, lane 3 is the RT-PCR amplification using primer sets of CH001-F and CH001-R, lane 4 RT-PCR amplification using a primer mixture consisting of CH001-F-high primer, CH001-R-high primer and high heel primer, lane 5 represents blank.

FIG. 5 shows the amplification of sample RNA by electrophoresis after RT-PCR was performed using the primer mixture according to the second embodiment of the present invention. Lane M is the size marker, lane 1 is the sample RNA itself, lane 2 is the PCR amplification of the sample RNA, lane 3 is the RT-PCR amplification using primer sets of CH003-F and CH003-R, lane 4 RT-PCR amplification using a primer mixture consisting of CH003-F-high primer, CH003-R-high primer and high heel primer, lane 5 represents blank.

Field of invention

The present invention relates to a probe composition and a primer for detecting a coronavirus and a method for detecting a coronavirus using the same, and more specifically, to a SARS virus using a leader sequence of a SARS virus and a base sequence encoding an N protein as targets for detection. It relates to a probe composition and a primer derived from a nucleotide sequence encoding a leader sequence or N protein of SARS virus designed to effectively detect the SARS virus and a method for effectively detecting SARS virus using the probe composition or the primer.

Background of the Invention

Severe Acute Respiratory Syndrome (SARS) has been developed since November 2002, mainly in Guangdong, China, and has fever, cough, shortness of breath, and atypical pneumonia, which has spread throughout the world including Hong Kong, Singapore, and Canada. to be. SARS is known to be caused by the variant SARS coronavirus.

The clinical symptoms of this viral infection are similar to those of common cold virus infections, but are deadly viruses that can be dangerous if not detected early.

In general, the following methods are used to analyze viruses.

The first is SSP-PCR, which uses primers specific for the HCV genotype. RT-PCR is performed by combining primers specific for several genotypes in the core region, and there is an advantage that results can be obtained immediately after RT-PCR. However, mutations occur in primer recognition sites or for new types. There is a disadvantage that it is difficult to detect.

Secondly, PCR-RFLP method (Park et al., J. Med. Microbiol. 47: 1998), which amplifies the 5'UTR site with RT-PCR and uses restriction enzymes, has a simple and easy result. However, if the restriction enzyme used does not recognize the mutant part, there is a disadvantage that the analysis is difficult.

Third, the 5'UTR site is amplified by RT-PCR and hybridized with a filter attached to an oligonucleotide probe specific to the genotype. This results in accurate results depending on the type of probe, but there are limitations in taking a large number of probes as a filter is used. Since only one person needs to be analyzed in one filter, it takes time and labor to process and analyze many samples. It is a lot of consumption.

SARS virus is a respiratory virus that contains about 29 kb of RNA as a genetic material, a variant of coronavirus, which produces about 6-10 subgenomic RNAs when infected with cells (FIG. 1).

As shown in FIG. 1, when a coronavirus is replicated in a cell, leader joining phenomenon occurs in which a leader sequence corresponding to 72 bp binds to the 5 ′ end of each subgenomic RNA. This leader sequence has the highest number of copies in the cell and the next highest number of copies of the subgenomic RNA encoding the N protein.

Therefore, for the effective detection of coronavirus, it is most effective to use the leader sequence and the N protein gene as targets for detection. These sequences are common to all known SARS viruses and therefore can detect all known SARS viruses.

The present inventors have devised a probe and a primer derived from a nucleotide sequence encoding the N protein and a leader sequence of the SARS virus, and confirmed that the SARS virus can be effectively detected by RT-PCR using the primer and the high heel primer. The invention was completed.

After all, the main object of the present invention is to provide a probe composition and primer for the detection of coronavirus including SARS virus, which is an infectious virus.

Another object of the present invention is to provide a method for effectively detecting a coronavirus including SARS virus using the probe composition or the primer.

In order to achieve the above object, the present invention provides a first embodiment, comprising: (a) a leader sequence of a SARS virus having a nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence complementary to (a) above; And (c) at least one nucleotide sequence selected from the group consisting of oligonucleotides having a continuous sequence of at least 10 bp derived from the nucleotide sequence of (a) or (b).

The first embodiment of the present invention also provides a method for detecting coronavirus, wherein the nucleic acid derived from a clinical sample of a suspected coronavirus infection is hybridized with the probe composition.

A first embodiment of the present invention also provides a coronavirus detection DNA chip, characterized in that the probe composition is integrated on a substrate and a method for detecting coronavirus, characterized in that using the DNA chip.

A first embodiment of the present invention also comprises: (a) an oligonucleotide having a contiguous sequence of 8 bp or more derived from a leader sequence of a SARS virus having the nucleotide sequence of SEQ ID NO: 1; And (b) a primer set for detecting coronaviruses containing an oligonucleotide having a contiguous sequence of 8 bp or more derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 1.

The first embodiment of the present invention also comprises: (a) a primer comprising an oligonucleotide having a consensus sequence of 8 bp or more derived from a leader sequence of a SARS virus having a nucleotide sequence of SEQ ID NO: 1 in a high heel primer; And (b) provides a primer set for detecting coronavirus containing a primer in combination with a high heel primer oligonucleotide having a sequence of at least 8bp derived from the sequence complementary to the base sequence of SEQ ID NO: 1. In the primer set, the oligonucleotide of (a) may have a nucleotide sequence of SEQ ID NO: 5, the oligonucleotide of (b) may be characterized by having a nucleotide sequence of SEQ ID NO: 4.

In a first embodiment of the present invention, RNA-extracted RNA from a clinical sample derived from a suspected coronavirus infection using the primer mixture and the primer mixture for detecting the coronavirus containing the primer set and the high heel primer is converted into RT-PCR. It provides a method for detecting coronavirus, characterized in that the amplification.

The present invention provides a second embodiment, (a) a base sequence encoding the N protein of the SARS virus represented by SEQ ID NO: 2; (b) a nucleotide sequence complementary to (a) above; And (c) at least one nucleotide sequence selected from the group consisting of oligonucleotides having a continuous sequence of at least 10 bp derived from the nucleotide sequence of (a) or (b).

A second embodiment of the present invention also provides a method for detecting coronavirus, characterized by hybridizing a nucleic acid derived from a clinical sample of a suspected coronavirus infection with the probe composition.

A second embodiment of the present invention also provides a coronavirus detection DNA chip, characterized in that the probe composition is integrated on a substrate and a method for detecting coronavirus, characterized in that using the DNA chip.

A second embodiment of the present invention also comprises: (a) an oligonucleotide having a contiguous sequence of 8 bp or more derived from the nucleotide sequence encoding the N protein of a SARS virus having the nucleotide sequence of SEQ ID NO: 2; And (b) a primer set for coronavirus detection containing an oligonucleotide having a contiguous sequence of 8 bp or more derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 2.

A second embodiment of the present invention also comprises: (a) a primer comprising an oligonucleotide having a contiguous sequence of 8 bp or more derived from a nucleotide sequence encoding an N protein of SARS virus represented by SEQ ID NO: 2 in a high heel primer; And (b) a primer set for coronavirus detection comprising a primer in which a high heel primer is combined with an oligonucleotide having a contiguous sequence of 8 bp or more derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 2. In the primer set, the oligonucleotide of (a) may have a nucleotide sequence of SEQ ID NO: 7, and the oligonucleotide of (b) may have a nucleotide sequence of SEQ ID NO: 6.

In a second embodiment of the present invention, the RNA mixture extracted from a clinical sample derived from a suspected coronavirus infection using the primer mixture and the primer mixture for detecting the coronavirus containing the primer set and the high heel primer was converted into RT-PCR. It provides a method for detecting coronavirus, characterized in that the amplification.

In the first and second embodiments of the present invention, the high heel primer may be characterized in that represented by SEQ ID NO: 3, coronavirus may be characterized in that the SARS virus, but is not limited thereto. In addition, the RT-PCR comprises the steps of (a) synthesizing cDNA from the RNA via reverse transcription; (b) synthesizing ds-cDNA using the synthesized cDNA as a template; And (c) amplifying the synthesized ds-cDNA using a high heel primer.

The term “high heel primer” used in the present invention is defined as an oligonucleotide having a Tm of about 72 ° C. and a GC content of 70% or more. The high heel primer is a primer designed to anneal at 65-75 ° C., preferably 72 ° C., to maximize the efficiency of linear amplification of RNA.

72 ° C. is also the temperature at which the efficiency of Taq polymerase commonly used for DNA amplification is maximized, thus optimizing the linear amplification process, and the annealing of the high heel primer and the extension of cDNA to be synthesized can occur simultaneously. The efficiency of time can be increased, thereby minimizing nonspecific amplification.

In the present invention, the high heel primer used is represented by SEQ ID NO: 3, but as defined above, as long as the Tm is about 72 ℃, GC content is 70% or more, but not limited thereto.

SEQ ID NO: 5'- CGC TGG GCC GAC CGG GCG CGG GAC-3 '

Embodiment 1: Specific Amplification of the SARS Virus Leader Sequence

Since SARS virus is an RNA virus, for amplification, a single-stranded cDNA complementary to the SARS virus sequence is first synthesized by reverse transcription. In this case, the reverse transcription primer uses a primer in which the SARS leader sequence specific primer represented by SEQ ID NO: 4 is combined with the high heel primer.

SEQ ID NO: 5'-gtt cgt tta gag aac aga tct a-3 '

In order to efficiently amplify the synthesized single stranded cDNA, SARS virus leader sequence, a PCR reaction is performed. The overall size of the SARS reader is 72bp, so it is difficult to confirm amplification in agarose gel electrophoresis. It is also important to increase the template annealing temperature in the amplification reaction to specifically amplify the SARS leader sequence.

Therefore, in order to facilitate specific amplification of SARS leader sequence and identification of amplification, a primer mixture including high heel primer is used in a PCR reaction (FIG. 2). Figure 2 is a schematic diagram showing the specific amplification of the SARS leader sequence according to the present invention.

Primer mixture according to the first embodiment of the present invention is as follows.

(1) high heel primer: the one represented by SEQ ID NO: 3 of 24 bp (more than 70 ° C.) having a high Tm value

(2) CH001-R-high primer: 22 bp CH001-R primer (SEQ ID NO: 4) corresponding to the 3 'end of the SARS virus leader sequence to the high heel primer of SEQ ID NO: 3.

(3) CH001-F-high primer: 22 bp CH001-F primer (SEQ ID NO: 5) corresponding to the 5 'end of the SARS virus leader sequence to the high heel primer of SEQ ID NO: 3

SEQ ID NO: 5'-ata tta ggt ttt tac cta ccc a-3 '

Embodiment 2: Specific Amplification of Sequence Encoding SARS Virus N Protein

Since SARS virus is an RNA virus, for amplification, a single-stranded cDNA complementary to the SARS virus sequence is first synthesized by reverse transcription. In this case, the reverse transcription primer uses a primer (CH003-R-high), which is a primer (SEQ ID NO: 6: CH003-R) specific to the nucleotide sequence encoding the N protein of the SARS virus, and a high heel primer.

SEQ ID NO: 5'-CTC TGC GTA GAA GCC TTT TGG-3 '

In order to effectively amplify the sequence encoding the synthesized single-stranded cDNA SARS virus N protein, a PCR reaction is performed. At this time, a primer was prepared to amplify a portion corresponding to about 188bp in the total size of SARS N protein of about 1.7kb. In addition, in order to specifically amplify the nucleotide sequence encoding the SARS N protein, the higher the annealing temperature between the template and the primer in the amplification reaction is advantageous.

Therefore, in order to facilitate identification of specific amplification and amplification of SARS, a primer mixture including high heel primers is used in a PCR reaction (FIG. 3). Figure 3 is a schematic diagram showing the specific amplification of the nucleotide sequence encoding SARS virus N protein using a high heel primer according to the present invention.

Primer mixture according to the second embodiment of the present invention is as follows.

(1) high heel primer: the one represented by SEQ ID NO: 3 of 24 bp (more than 70 ° C.) having a high Tm value

(2) CH003-R-high primer: The high heel primer of SEQ ID NO: 3 is linked to a 21 bp CH003-R primer (SEQ ID NO: 6) corresponding to 3 'of the nucleotide sequence encoding the SARS virus N protein.

(3) CH003-F-high primer: The high heel primer of SEQ ID NO: 3 is linked to a 21 bp CH003-F primer (SEQ ID NO: 7) corresponding to 5 'of the nucleotide sequence encoding the SARS virus N protein.

SEQ ID NO: 5'-ACCTAGGAACT GGCCCAGAAG-3 '

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

In particular, the following examples exemplify a method for detecting coronavirus by RT-PCR using primers derived from a leader sequence of the SARS virus and a nucleotide sequence encoding the N protein and a high heel primer. It will be apparent to those skilled in the art to design probe compositions using sequences and to detect coronaviruses by hybridizing nucleic acids derived from clinical samples with the probe compositions. In addition, it will be apparent to those skilled in the art that the probe composition may be integrated on a substrate to produce a DNA chip, and to detect coronaviruses using the same.

Example 1: Specific Amplification of SARS Virus Leader Sequence

(1) Synthesis of single stranded cDNA by reverse transcription

Total RNA was extracted from clinical samples of suspected SARS virus infection (Invitrogen, Trizol method). 1-5 μl of extracted sample RNA was annealed by incubating 1 μl of CH001-R-high primer (2 μg / μl) at 65 ° C. for 10 minutes. The reaction volume was 15.4 μl at the time of annealing. To the annealed reaction solution, 6 µl of 5X fisrt strand buffer (Invitrogen), 3 µl of 0.1 mM DTT (Invitrogen), 0.6 µl of 25 mM dNTP (Amersham), and 1 µl of reverse transcriptase (SuperscriptII, 200U, Invitrogen) were added. The final reaction solution was 25 μl and then incubated at 42 ° C. for 2 hours.

(2) Amplification of SARS Leader Sequence

PCR amplification of the SARS leader sequence was designed to be performed in one tube. PCR was performed using the high heel primer of SEQ ID NO: 3, the CH001-R-high primer and the CH001-F-high primer at the same time, and using the single-stranded leader sequence cDNA made by reverse transcription as a template. That is, 2.5 μl of 10X PCR buffer (Solgent, Korea), 1 μl of Taq polymerase (5U / μl, Solgent, Korea), 1 μl of dNTP (2.5 mM, Solgent, Korea) in 2.5 μl of the reverse transcription mixture prepared in (1) above. 1 μl of CH001-F-high primer (10 pmole / μl), 1 μl of CH001-R-high primer (10 pmole / μl) and 1 μl of high heel primer (100 pmole / μl) of SEQ ID NO. 1 μl of PCR [10 min at 94 ° C., 1 min at 94 ° C., 1 min at 50 ° C., 30 sec at 72 ° C.) was performed. During PCR, a template necessary for secondary PCR is made by CH001-R-high and CH001-F-high primers.

Thereafter, the amplification reaction was performed using a high heel primer of SEQ ID NO. 3 (1 minute at 94 ° C., 45 seconds at 72 ° C.) and 35 to 40 cycles, and final extension at 72 ° C. for 10 minutes. In the amplification reaction, an amplification product of about 120 bp comprising the SARS leader sequence (72 bp) is specifically made. Electrophoresis was performed to confirm that the amplification products were made specifically (FIG. 4).

Figure 4 is a picture of the electrophoresis on 5% of a total of 25μl reaction solution on 1.5% agarose gel. Lane M is the size marker, lane 1 is the sample RNA itself, lane 2 is the PCR amplification of the sample RNA, lane 3 is the RT-PCR amplification using primer sets of CH001-F and CH001-R, lane 4 RT-PCR amplification using a primer mixture consisting of CH001-F-high primer, CH001-R-high primer and high heel primer, lane 5 represents blank.

As shown in FIG. 4, if the RNA itself (lane 1) or RNA itself is PCR (lane 2), no PCR product is produced, but a primer set of CH001-F and CH001-R is used, or CH001-R- When RT-PCR was performed using a primer mixture consisting of a high primer, a CH001-F-high primer, and a high heel primer, SARS leader sequences of about 72 bp and about 120 bp, respectively, were specifically amplified, in particular high heels primers were added. In one case, the amplification efficiency was better.

As a result, in this embodiment, by increasing the annealing temperature of the template and primer during amplification, it can be confirmed that it is possible to minimize non-specific amplification and to specifically amplify the SARS leader sequence.

Example 2: Specific Amplification of Sequence Encoding SARS Virus N Protein

(1) Synthesis of single stranded cDNA by reverse transcription

1-5 μl of sample RNA extracted from clinical samples of suspected SARS virus infection was annealed by incubating 1 μl of CH003-R-high primer (2 μg / μl) at 65 ° C. for 10 minutes. The reaction volume was 15.4 μl at the time of annealing. To the annealed reaction solution, 6 µl of 5X fisrt strand buffer (Invitrogen), 3 µl of 0.1 mM DTT (Invitrogen), 0.6 µl of 25 mM dNTP (Amersham), and 1 µl of reverse transcriptase (SuperscriptII, 200U, Invitrogen) were added. The final reaction solution was 25 μl and then incubated at 42 ° C. for 2 hours.

(2) Amplification of the nucleotide sequence encoding the SARS N protein

The nucleotide sequence encoding the N protein was specifically amplified by a method similar to the PCR amplification method of the SARS leader sequence of Example 1, and the amplification reaction was designed to be performed in one tube. The high heel primer of SEQ ID NO: 3, CH003-R-high primer and CH003-F-high primer were put at the same time, and PCR was performed using a single-stranded leader sequence cDNA made by reverse transcription as a template. That is, 2.5 μl of 10X PCR buffer (Solgent, Korea), 1 μl of Taq polymerase (5U / μl, Solgent, Korea), 1 μl of dNTP (2.5 mM, Solgent, Korea) in 2.5 μl of the reverse transcription mixture prepared in (1) above. 1 μl of CH003-F-high primer (10 pmole / μl), 1 μl of CH003-R-high primer (10 pmole / μl) and 1 μl of high heel primer (100 pmole / μl) of SEQ ID NO. 1 μl of PCR (10 min at 94 ° C., 1 min at 94 ° C., 1 min at 50 ° C., 30 sec at 72 ° C.) was performed. During PCR, a template necessary for secondary PCR is made by using CH003-R-high and CH003-F-high primers.

Thereafter, the amplification reaction was performed using a high heel primer of SEQ ID NO. 3 (1 minute at 94 ° C., 45 seconds at 72 ° C.) and 35 to 40 cycles, and 10 minutes at 72 ° C. as a final extension. In the amplification reaction, an amplification product of about 236 bp comprising a nucleotide sequence encoding about SARS N protein (about 188 bp) is specifically made. In order to confirm that the amplification product is made specifically, electrophoresis was performed (FIG. 5).

5 is a photograph of 5 μl of a total of 25 μl of reaction solution electrophoresed on 1.5% agarose gel. In FIG. 5, lane M is a size marker, lane 1 is sample RNA itself, lane 2 is PCR amplification of sample RNA, and lane 3 is RT-PCR amplification using primer sets of CH003-F and CH003-R. , Lane 4 represents the RT-PCR amplification using a primer mixture consisting of CH003-F-high primer, CH003-R-high primer and high heel primer, lane 5 represents blank.

As shown in FIG. 5, when the RNA itself (lane 1) or RNA itself is PCR (lane 2), PCR products are not produced, but primer sets of CH003-F and CH003-R are used, or CH003-R- When RT-PCR was performed using a primer mixture consisting of a high primer, a CH003-F-high primer, and a high heel primer, the nucleotide sequences encoding SARS N protein of about 188 bp and about 236 bp, respectively, were specifically amplified. In particular, the amplification efficiency was better when the high heel primer was added.

As a result, in this embodiment, by increasing the annealing temperature of the template and primer during amplification, it was confirmed that non-specific amplification can be minimized, and it is possible to specifically amplify the nucleotide sequence encoding the SARS N protein.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do.

The RT-PCR system using the primer mixture for SARS virus detection according to the present invention can specifically amplify only the nucleotide sequence encoding the SARS virus leader or N protein, thereby effectively detecting the coronavirus containing SARS virus. Useful for

In addition, since virus titers are generally very low in clinical samples, two or more nested PCRs should be performed in the prior art. In the present invention, four heels primers are used in the final amplification reaction. It is expected to replace the effects of sted PCR.

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Claims (26)

(a) a leader sequence of a SARS virus having the nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence complementary to (a) above; And (c) at least one nucleotide sequence selected from the group consisting of oligonucleotides having a contiguous sequence of at least 10 bp derived from the leader sequence of (a) or the complementary nucleotide sequence of (b). Probe composition. A method for detecting coronavirus, comprising hybridizing a nucleic acid derived from a clinical sample of a suspected coronavirus infection with a probe composition of claim 1. A DNA chip for corona virus detection, wherein the probe composition of claim 1 is integrated on a substrate. A method for detecting coronavirus, comprising using the DNA chip of claim 3. (a) an oligonucleotide having a continuous sequence of 8 to 30 bp derived from a leader sequence of a SARS virus having the nucleotide sequence of SEQ ID NO: 1; And (b) a primer set for coronavirus detection comprising oligonucleotides in a sequence of 8 to 30 bp consecutive sequences derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 1. (a) a primer linked to an oligonucleotide having a contiguous sequence of 8 to 30 bp derived from a leader sequence of a SARS virus having a nucleotide sequence of SEQ ID NO: 1 at the 3 'end of the high heel primer of SEQ ID NO: 3; And (b) a primer connected to an oligonucleotide having a contiguous sequence of 8 to 30 bp derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 at the 3 'end of the high heel primer of SEQ ID NO: 3 Primer set. The corona according to claim 6, wherein the oligonucleotide constituting the primer (a) has a nucleotide sequence of SEQ ID NO: 5, and the oligonucleotide constituting the (b) primer has a nucleotide sequence of SEQ ID NO: 4 Primer set for virus detection. The primer set according to claim 6, wherein the high heel primer is represented by SEQ ID NO. Primer mixture for detecting a coronavirus containing a primer set of any one of claims 6 to 8. 10. The primer mixture of claim 9, wherein the high heel primer is represented by SEQ ID NO: 3. A method for detecting coronavirus, comprising amplifying RNA extracted from a clinical sample derived from a suspected coronavirus infection by RT-PCR using the primer mixture of claim 9. The method of claim 11, wherein the RT-PCR is subjected to the following steps: (a) synthesizing cDNA from the RNA via reverse transcription; (b) synthesizing ds-cDNA using the synthesized cDNA as a template; And (c) using the synthesized ds-cDNA as a template and amplifying using the primer mixture of claim 9. The method of claim 11, wherein the coronavirus is a SARS virus. delete delete delete delete delete (a) a primer linked to an oligonucleotide having a contiguous sequence of 8 to 30 bp derived from a nucleotide sequence encoding the N protein of the SARS virus represented by SEQ ID NO: 2 at the 3 'end of the high heel primer of SEQ ID NO: 3; And (b) a primer connected to an oligonucleotide having a contiguous sequence of 8 to 30 bp derived from a sequence complementary to the nucleotide sequence of SEQ ID NO: 2 at the 3 'end of the high heel primer of SEQ ID NO: 3 Primer set. The corona according to claim 19, wherein the oligonucleotide constituting the primer (a) has a nucleotide sequence of SEQ ID NO: 7, and the oligonucleotide constituting the (b) primer has a nucleotide sequence of SEQ ID NO: 6 Primer set for virus detection. delete Primer mixture for detection of coronavirus containing the primer set of claim 19 or 20. delete A method for detecting coronavirus, comprising amplifying RNA extracted from clinical samples derived from suspected coronavirus infection by RT-PCR using the primer mixture of claim 22. The method of claim 24, wherein the RT-PCR is subjected to the following steps: (a) synthesizing cDNA from the RNA via reverse transcription; (b) synthesizing ds-cDNA using the synthesized cDNA as a template; And (c) using the synthesized ds-cDNA as a template and amplifying using the primer mixture of claim 24. The method of claim 24, wherein the coronavirus is a SARS virus.

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