PCR PRIMER SET DETECTING SEVERE ACUTE RESPIRATORY SYNDROME (SARS)-CORONAVIRUS, METHOD AND KIT FOR DETECTING SARS-CORONAVIRUS USING THE SAME

Info

Publication number: 20080194422
Type: Application
Filed: Apr 21, 2008
Publication Date: Aug 14, 2008
Applicant: SAMSUNG ELECTRONICS CO., LTD. (Suwon-si)
Inventors: Hee-kyun LIM (Suwon-si), Sang-hyo KIM (Yongin-si), Jung-joo HWANG (Suwon-si), Young-sun LEE (Seongnam-si), Young-a KIM (Suwon-si)
Application Number: 12/106,686

Abstract

Provided are SARS-CoV detection PCR primers having nucleotide sequences as set forth in SEQ ID NOS: 1-46, a method for detecting SARS-CoV using the primers, and a SARS-CoV detection kit including the primers.

Description

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. patent application Ser. No. 10/997,462, filed Nov. 24, 2004, which claims the benefit of Korean Patent Application No. 2003-90559, filed on Dec. 12, 2003, in the Korean Intellectual Property Office, the disclosure of each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to PCR primer sets for detecting severe acute respiratory syndrome (SARS) virus, a method and a kit for detecting SARS virus.

2. Description of the Related Art

Severe acute respiratory syndrome (SARS) virus is a new type of coronavirus and is designated as SARS-Coronavirus (hereinafter, referred to as “SARS-CoV”) by the World Health Organization (WHO). It is known that SARS-CoV gives rise to severe acute respiratory syndrome. SARS-COV is a novel RNA virus. It is known that the whole genome sequence of SARS-COV consists of about 29,730 bp and the genetic makeup is similar to those of other types of coronaviruses.

The WHO reported guidelines for laboratory diagnosis of SARS as follows: (a) antibody test: identification of SARS-CoV antibody in serum after one day, (b) PCR (Polymerase Chain Reaction) test: identification of SARS-COV RNA by PCR, and (c) virus separation: separation of SARS-CoV.

In a conventional PCR test, the following six types of primers are used for detection of SARS-COV:

- (1) BNIoutS2: SEQ ID NO: 29 (24 mer); BNIoutAs: SEQ ID NO: 30 (21 mer), BNIinS: SEQ ID NO: 31 (20 mer); BNIinAs: SEQ ID NO: 32 (22 mer),
- (2) SAR1S: SEQ ID NO: 33 (21 mer); SAR1As: SEQ ID NO: 34 (21 mer),
- (3) Cor-p-F2: SEQ ID NO: 35 (21 mer); Cor-p-R1: SEQ ID NO: 34 (21 mer),
- (4) Cor-p-F3: SEQ ID NO: 35 (21 mer); Cor-p-R1: SEQ ID NO: 36 (21 mer),
- (5) COR-1: SEQ ID NO: 37 (26 mer); COR-2: SEQ ID NO: 38 (26 mer),
- (6) HKU (sense): SEQ ID NO: 39 (17 mer); HKU (antisense): SEQ ID NO: 40 (16 mer).

Among them, the primers of (1), which were developed by Bernhard-Nocht Institute (BNI, Germany) for diagnosis of SARS generated in early 2003, have been most widely used. However, only a local region, i.e., a polymerase gene region, of the whole genome sequence of about 30 kb of SARS-COV, is used as a target site of currently known primers. Such a narrow application site may lead to either false negative or false positive results.

In view of these problems, the present inventors found SARS-CoV-specific primers which reduce a likelihood of being determined as false negative or false positive and rapidly detect SARS-COV in situ after rapid PCR, and completed the present invention.

SUMMARY OF THE INVENTION

The present invention provides SARS-CoV-specific primer sets.

The present invention also provides a method for specifically detecting SARS-COV using the primer sets.

The present invention also provides a SARS-COV detection kit including the primer set.

According to an aspect of the present invention, there is provided a PCR primer set for SARS-COV detection selected from the group consisting of the following primer sets: (a) a primer set (GS-SARS01) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 1 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 2; (b) a primer set (GS-SARS02) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 4; (c) a primer set (GS-SARS03) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 5 and a primer having a nucleotide primer having a nucleotide sequence as set forth in SEQ ID NO: 35 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 36; (s) a primer set (GS-SARS19) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 37 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 38; (t) a primer set (GS-SARS20) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 39 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 40; (u) a primer set (GS-SARS21) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 41 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 42; (v) a primer set (GS-SARS22) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 43 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 44; and (w) a primer set (GS-SARS23) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 45 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 46.

According to another aspect of the present invention, there is provided a method for detecting SARS-COV, which includes amplifying a nucleic acid sample obtained from an individual by PCR using the primer set.

According to yet another aspect of the present invention, there is provided a SARS-COV detection kit including the primer set.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 schematically illustrates target nucleotide sequences for PCR primers according to the present invention;

FIG. 2 illustrates tube RT-PCR results for SARS-COV RNA samples using primers according to the present invention;

FIG. 3 illustrates PCR results for SARS-COV RNA samples on a silicon-based PCR chip using primers according to the present invention; and

FIG. 4 illustrates PCR results for SARS-COV RNA samples and human gDNA samples on a silicon-based PCR chip using primers according to the present invention. sequence as set forth in SEQ ID NO: 6; (d) a primer set (GS-SARS04) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 7 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 8; (e) a primer set (GS-SARS05) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 9 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 10; (f) a primer set (GS-SARS06) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 11 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 12; (g) a primer set (GS-SARS07) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 13 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 14; (h) a primer set (GS-SARS08) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 15 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 16; (i) a primer set (GS-SARS09) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 17 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 18; (j) a primer set (GS-SARS10) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 19 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 20; (k) a primer set (GS-SARS11) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 21 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 22; (l) a primer set (GS-SARS12) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 23 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 24; (m) a primer set (GS-SARS13) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 25 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 26; (n) a primer set (GS-SARS14) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 27 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 28; (o) a primer set (GS-SARS15) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 29 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 30; (p) a primer set (GS-SARS16) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 31 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 32; (q) a primer set (GS-SARS17) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 33 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 34; (r) a primer set (GS-SARS18) including a

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a PCR primer set selected from the group consisting of the following primer sets:

(a) a primer set (GS-SARS01) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 1 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 2;

(b) a primer set (GS-SARS02) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 4;

(c) a primer set (GS-SARS03) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 5 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 6;

(d) a primer set (GS-SARS04) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 7 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 8;

(e) a primer set (GS-SARS05) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 9 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 10;

(f) a primer set (GS-SARS06) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 11 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 12;

(g) a primer set (GS-SARS07) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 13 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 14;

(h) a primer set (GS-SARS08) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 15 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 16;

(i) a primer set (GS-SARS09) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 17 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 18; as set forth in SEQ ID NO: 39 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 40;

(u) a primer set (GS-SARS21) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 41 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 42;

(v) a primer set (GS-SARS22) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 43 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 44; and

(w) a primer set (GS-SARS23) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 45 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 46.

The primer sets of the present invention can specifically detect SARS-COV without reacting with other coronaviruses. That is, when PCR is performed using the primer set of the present invention, PCR products are obtained from individuals infected with SARS-COV but no PCR products are obtained from individuals infected with other coronaviruses. The PCR primer set for SARS-COV detection of the present invention is_selected from a non-structural region and a structural region among the genome sequence of SARS-CoV. SARS-COV regions including target nucleotide sequences for the primers according to the present invention are illustrated in FIG. 1.

The present invention also provides a method for detecting SARS-COV, which comprises amplifying a nucleic acid sample obtained from an individual by PCR using the primer set for SARS-CoV detection.

As used herein, the term “PCR” is well known in the pertinent art. Generally, PCR includes the steps of: (a) obtaining a crude extract containing target DNA molecules from a sample; (b) adding an aqueous solution including an enzyme, a buffer, dNTPs, and oligonucleotide primers to the crude extract; (c) amplifying the target DNA molecules by two- or three-step thermal cycling (e.g., 90-96° C., 72° C., and 37-55° C.) of the resultant mixture; and (d) detecting amplified DNAs.

In the present invention, the PCR may be performed in a polypropylene tube, a 96-well plate, or a silicon-based micro PCR chip.

When the PCR is performed on a silicon-based micro PCR chip, a two-step thermal cycling as well as a three-step thermal cycling can be used. A time required for the PCR on the silicon-based micro PCR chip can be as short as 30 minutes or less. For example, the silicon-based micro PCR chip includes a silicon wafer, a surface of which is formed with a PCR chamber made by silicon lithography and the other surface is formed with a heater for heating the PCR chamber; and a glass wafer having an inlet and an outlet.

In the present invention, the PCR may be performed using 0.2-1 μM of each primer and 0.01 pg to 1 μg of a template DNA.

In the present invention, the PCR may be performed in three-step thermal cycling conditions of denaturation at 86-97° C. for 1-30 seconds, annealing at 50-70° C. for 1-30 seconds, and extension at 60-72° C. for 1-30 seconds, or in two-step thermal cycling conditions of denaturation at 86-97° C. for 1-30 seconds and annealing and extension at 50-70° C. for 5-30 seconds.

The present invention also provides a SARS-CoV detection kit including the primer set for SARS-CoV detection.

The SARS-COV detection kit of the present invention may include the primer set, a PCR solution, a buffer, an enzyme, and the like.

Hereinafter, the present invention will be described more specifically by Examples. However, the following Examples are provided only for illustrations and thus the present invention is not limited to or by them.

EXAMPLES Example 1 Design of Primers for SARS-CoV Detection

In this Example, there were designed PCR primers for SARS-COV detection, which are specific to SARS-CoVs and do not react with other coronaviruses.

The genome sequences of 40 strains belonging to SARS-CoVs were searched using the public database (www.ncbi.nim.nih.gov). Based on the search results, common sequences of the 40 strains were extracted. After sequence homology search between the common sequences and other coronaviruses and other respiratory viruses capable of infecting human bodies, homologous sequences were removed and sequences with no cross reactivity were selected. The results are presented in Table 1 below.

TABLE 1 Analysis results for cross reactivity using B12Seq or Blastn H_CoV Human Section H_CoV229E OC43 INFV PINFV RSV ADV HMPV gDNA Sequence NS* NS NS NS NS NS NS PM** matching *NS: No significant homology **PM: Partially matching H_CoV229E: Human coronavirus 229E, H_CoV OC43: Human coronavirus OC43 INFV: Influenza virus type A, B, PINFV: Parainfluenza virus type 1, 2, 3 RSV: Respiratory syncytial virus type A, B ADV: Adenovirus, HMPV: Human metapneumovirus

As a result, primers as listed in Table 2 below were selected.

(j) a primer set (GS-SARS10) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 19 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 20;

(k) a primer set (GS-SARS11) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 21 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 22;

(l) a primer set (GS-SARS12) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 23 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 24;

(m) a primer set (GS-SARS13) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 25 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 26;

(n) a primer set (GS-SARS14) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 27 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 28;

(o) a primer set (GS-SARS15) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 29 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 30;

(p) a primer set (GS-SARS16) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 31 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 32;

(q) a primer set (GS-SARS17) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 33 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 34;

(r) a primer set (GS-SARS18) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 35 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 36;

(s) a primer set (GS-SARS19) including a primer having a nucleotide sequence as set forth in SEQ ID NO: 37 and a primer having a nucleotide sequence as set forth in SEQ ID NO: 38;

(t) a primer set (GS-SARS20) including a primer having a nucleotide sequence

TABLE 2 PCR primers for SARS-CoV detection and their characteristics Primer Set Primer No. No. Sequence Position Length Tm (° C.) PCR product (bp) GS-SARS01 A1F4 SEQ ID NO: 1 3196-3221 26 58.6 156 A1R4 SEQ ID NO: 2 3328-3351 24 58.4 GS-SARS02 C30F4 SEQ ID NO: 3 10443-10467 25 57.1 244 C30R4 SEQ ID NO: 4 10662-10686 25 59.7 GS-SARS03 FJF3 SEQ ID NO: 5 20594-20615 22 64.1 266 FJR3 SEQ ID NO: 6 20836-20859 24 63.8 GS-SARS04 S2F1 SEQ ID NO: 7 24097-24121 25 70.6 118 S2R1 SEQ ID NO: 8 24187-24213 27 67.8 GS-SARS05 CMF2 SEQ ID NO: 9 26429-26454 26 60.9 179 CMR2 SEQ ID NO: 10 26584-26607 24 59.1 GS-SARS06 CNF2 SEQ ID NO: 11 28560-28585 26 66.3 103 CNR2 SEQ ID NO: 12 28640-28662 23 67.5 GS-SARS07 A1F3 SEQ ID NO: 13 3236-3259 24 59.2 184 A1R3 SEQ ID NO: 14 3398-3419 22 61.3 GS-SARS08 C30F3 SEQ ID NO: 15 10443-10467 25 57.1 242 C30R3 SEQ ID NO: 16 10659-10684 26 56.0 GS-SARS09 FJF2 SEQ ID NO: 17 20590-20615 26 68.6 275 FJR2 SEQ ID NO: 18 20839-20864 26 69.5 GS-SARS10 S2F3 SEQ ID NO: 19 24194-24216 23 61.7 218 S2R3 SEQ ID NO: 20 24388-24411 24 60.5 GS-SARS11 S2F4 SEQ ID NO: 21 24044-24068 25 61.8 167 S2R4 SEQ ID NO: 22 24187-24210 24 61.4 GS-SARS12 CMF1 SEQ ID NO: 23 26429-26454 26 60.9 275 CMR1 SEQ ID NO: 24 26677-26703 27 64.4 GS-SARS13 CNF4 SEQ ID NO: 25 28643-28666 24 68.4 145 CNR4 SEQ ID NO: 26 28764-28787 24 67.3 GS-SARS14 CNF5 SEQ ID NO: 27 28645-28666 22 64.9 140 CNR5 SEQ ID NO: 28 28764-28784 21 66.5 GS-SARS15 A1F1 SEQ ID NO: 29 3156-3179 24 65.1 267 A1R1 SEQ ID NO: 30 3398-3422 25 65.4 GS-SARS16 A1F2 SEQ ID NO: 31 3231-3256 26 60.7 121 A1R2 SEQ ID NO: 32 3327-3351 25 60.9 GS-SARS17 C30F1 SEQ ID NO: 33 10441-10467 27 59.2 246 C30R1 SEQ ID NO: 34 10660-10686 27 61.2 GS-SARS18 C30F2 SEQ ID NO: 35 10190-10214 25 64.7 102 C30R2 SEQ ID NO: 36 10265-10291 27 55.2 GS-SARS19 FJF1 SEQ ID NO: 37 20701-20728 28 60.5 115 FJR1 SEQ ID NO: 38 20794-20815 22 56.2 GS-SARS20 S2F2 SEQ ID NO: 39 24042-24068 27 64.2 175 S2R2 SEQ ID NO: 40 24191-24216 26 64.8 GS-SARS21 S2F5 SEQ ID NO: 41 24492-24516 25 61.8 123 S2R5 SEQ ID NO: 42 24595-24614 20 65.5 GS-SARS22 CNF1 SEQ ID NO: 43 28641-28666 26 70.2 151 CNR1 SEQ ID NO: 44 28766-28791 26 70.0 GS-SARS23 CNF3 SEQ ID NO: 45 28640-28666 27 72.1 150 CNR3 SEQ ID NO: 46 28764-28789 26 71.9

Example 2 Tube RT-PCR for SARS-CoV RNA Samples

In this Example, RT-PCR was performed on a PCR machine (ABI 2400) using the primer sets of GS-SARS01-06 and SARS-CoV RNA (total RNA samples extracted from SARS-CoVs) as templates. At this time, the concentration of the templates was 2 μg/20 μl. Before initiating PCR, the RNA samples in a 25 μl PCR solution were converted to cDNAs by reverse transcription at 42° C. for 60 minutes. The PCR was performed in three-step thermal cycling conditions as follows: 50 cycles of denaturation at 95° C. for 20 seconds, annealing 65° C. for 40 seconds, and extension at 72° C. for 60 seconds.

The results are shown in FIG. 2. As shown in FIG. 2, SARS-CoV RNAs were detected. In FIG. 2, lane 1 is 1 kb-DNA plus ladder (Invitrogen), lane 2 is a PCR product (156 bp) amplified with GS-SARS01, lane 3 is a PCR product (244 bp) amplified with GS-SARS02, lane 4 is a PCR product (179 bp) amplified with GS-SARS05, lane 5 is a PCR product (103 bp) amplified with GS-SARS06, and lane 6 is a PCR product (118 bp) amplified with GS-SARS04.

Example 3 RT-PCR for SARS-CoV RNA Samples on Silicon-Based PCR Chip

In this Example, RT-PCR was performed on a PCR machine (GenSpector 1&2) using the primer sets of GS-SARS01-06 and SARS-COV RNAs (total RNA samples extracted from SARS-CoVs) as templates. At this time, the concentration of the templates was 2.4×10³copies. Before initiating PCR, the RNA samples in a 1 μl PCR solution on a silicon-based PCR chip were converted to cDNAs by reverse transcription at 42° C. for 60 minutes. The PCR was performed in three-step thermal cycling conditions as follows: 50 cycles of denaturation at 92° C. for 1 second, annealing 52° C. for 1 second, and extension at 72° C. for 5 seconds.

The results are shown in FIG. 3. As shown in FIG. 3, SARS-COV RNAs were detected on the silicon-based PCR chip. Each lane of FIG. 3 is as defined in FIG. 2.

Example 4 Cross Reactivity Test With Human gDNAs

In this Example, whether the primer sets of GS-SARS01-14 react with human gDNAs was analyzed.

PCR was performed in the same manner as in Example 3 except that 45 cycles were repeated using a 25 μl PCR solution containing 1 ng SARS-COV cDNAs+200 ng human gDNAs as templates.

The results are shown in FIG. 4. As shown in FIG. 4, no cross reactivity with human gDNAs were observed and SARS-COV RNAs were detected. In FIG. 4, M is 1 kb-DNA plus ladder (Invitrogen), lane 1 is a PCR product (184 bp) amplified with GS-SARS07, lane 2 is a PCR product (156 bp) amplified with GS-SARS01, lane 3 is a PCR product (275 bp) amplified with GS-SARS09, lane 4 is a PCR product (266 bp) amplified with GS-SARS03, lane 5 is a PCR product (242 bp) amplified with GS-SARS08, lane 6 is a PCR product (244 bp) amplified with GS-SARS02, lane 7 is a PCR product (275 bp) amplified with GS-SARS12, lane 8 is a PCR product (179 bp) amplified with GS-SARS05, lane 9 is a PCR product (103 bp) amplified with GS-SARS03, lane 10 is a PCR product (145 bp) amplified with GS-SARS13, lane 11 is a PCR product (140 bp) amplified with GS-SARS14, lane 12 is a PCR product (118 bp) amplified with GS-SARS04, lane 13 is a PCR product (218 bp) amplified with GS-SARS10, and lane 14 is a PCR product (167 bp) amplified with GS-SARS11.

As apparent from the above description, a primer set of the present invention can be efficiently used in a method for specifically detecting SARS-COV.

A method for detecting SARS-CoV of the present invention can detect SARS-COV without cross reaction of the primer set with other coronaviruses.

A SARS-COV detection kit of the present invention can be used in specifically detecting SARS-COV.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A PCR primer set selected from the group consisting of the following primer sets:

(a) a primer set (GS-SARS02) comprising a primer consisting of SEQ ID NO: 3 and a primer consisting of SEQ ID NO: 4;

(b) a primer set (GS-SARS03) comprising a primer consisting of SEQ ID NO: 5 and a primer consisting of SEQ ID NO: 6;

(b) a primer set (GS-SARS04) comprising a primer consisting of SEQ ID NO: 7 and a primer consisting of SEQ ID NO: 8;

(c) a primer set (GS-SARS05) comprising a primer consisting of SEQ ID NO: 9 and a primer consisting of SEQ ID NO: 10;

(d) a primer set (GS-SARS06) comprising a primer consisting of SEQ ID NO: 11 and a primer consisting of SEQ ID NO: 12;

(e) a primer set (GS-SARS07) comprising a primer consisting of SEQ ID NO: 13 and a primer consisting of SEQ ID NO: 14;

(f) a primer set (GS-SARS08) comprising a primer consisting of SEQ ID NO: 15 and a primer consisting of SEQ ID NO: 16;

(g) a primer set (GS-SARS09) comprising a primer consisting of SEQ ID NO: 17 and a primer consisting of SEQ ID NO: 18;

(h) a primer set (GS-SARS10) comprising a primer consisting of SEQ ID NO: 19 and a primer consisting of SEQ ID NO: 20;

(i) a primer set (GS-SARS11) comprising a primer consisting of SEQ ID NO: 21 and a primer consisting of SEQ ID NO: 22;

(j) a primer set (GS-SARS12) comprising a primer consisting of SEQ ID NO: 23 and a primer consisting of SEQ ID NO: 24;

(k) a primer set (GS-SARS13) comprising a primer consisting of a SEQ ID NO: and a primer consisting of SEQ ID NO: 26;

(l) a primer set (GS-SARS14) comprising a primer consisting of as set forth in SEQ ID NO: 27 and a primer consisting of as set forth in SEQ ID NO: 28;

(m) a primer set (GS-SARS15) comprising a primer consisting of SEQ ID NO: 29 and a primer consisting of SEQ ID NO: 30;

(n) a primer set (GS-SARS16) comprising a primer consisting of SEQ ID NO: 31 and a primer consisting of SEQ ID NO: 32;

(o) a primer set (GS-SARS17) comprising a primer consisting of SEQ ID NO: 33 and a primer consisting of SEQ ID NO: 34;

(q) a primer set (GS-SARS18) comprising a primer consisting of SEQ ID NO: 35 and a primer consisting of SEQ ID NO: 36;

(r) a primer set (GS-SARS19) comprising a primer consisting of SEQ ID NO: 37 and a primer consisting of SEQ ID NO: 38;

(s) a primer set (GS-SARS20) comprising a primer consisting of SEQ ID NO: 39 and a primer consisting of SEQ ID NO: 40;

(t) a primer set (GS-SARS21) comprising a primer consisting of SEQ ID NO: 41 and a primer consisting of SEQ ID NO: 42;

(u) a primer set (GS-SARS22) comprising a primer consisting of SEQ ID NO: 43 and a primer consisting of SEQ ID NO: 44; and

(v) a primer set (GS-SARS23) comprising a primer consisting of SEQ ID NO: 45 and a primer consisting of SEQ ID NO: 46,

wherein the primer set specifically amplifies a target region of Severe Acute Respiratory syndrome corona virus (SARS-CoV) in a polymerase chain reaction (PCR) and the primers do not share sequence homology with human coronavirus 229E; human coronavirus OC43; influenza virus type A; influenza virus type B; parainfluenza virus types 1, 2 or 3; respiratory syncytial virus type A, respiratory syncytial virus type B; or human metapneumovirus.

2. A method for detecting SARS-COV, which comprises amplifying a nucleic acid sample obtained from an individual by PCR using the primer set of claim 1.

3. The method of claim 2, wherein the PCR is performed in a polypropylene tube, a 96-well plate, or a silicon-based micro PCR chip.

4. The method of claim 2, wherein the PCR is a two-step PCR comprising a denaturation step and an annealing and extension step or a three-step PCR comprising a denaturation step, an annealing step, and an extension step.

5. The method of claim 2, wherein the PCR is performed using 0.2-1 μM of each primer and 0.01 pg to 1 μg of a template DNA.

6. The method of claim 2, wherein a time required for the PCR on a silicon-based micro PCR chip is 30 minutes or less.

7. The method of claim 2, wherein the PCR is performed in three-step thermal cycling conditions of denaturation at 86-97° C. for 1-30 seconds, annealing at 50-70° C. for 1-30 seconds, and extension at 60-72° C. for 1-30 seconds, or in two-step thermal cycling conditions of denaturation at 86-97° C. for 1-30 seconds and annealing and extension at 50-70° C. for 5-30 seconds.

8. The method of claim 6, wherein the silicon-based micro PCR chip comprises:

a silicon substrate, a surface of which is formed with a PCR chamber made by silicon lithography and the other surface is formed with a heater for heating the PCR chamber; and

a glass substrate having an inlet and an outlet.

9. A SARS-COV detection kit comprising the primer set of claim 1.

10. The primer set of claim 1, wherein the primers do not share sequence homology with each of human coronavirus 229E, human coronavirus OC43, influenza virus type A, influenza virus type B; parainfluenza virus types 1, 2, and 3; respiratory syncytial virus types A and B; and human metapneumovirus.