US20080194422A1 - Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same - Google Patents

Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same Download PDF

Info

Publication number
US20080194422A1
US20080194422A1 US12/106,686 US10668608A US2008194422A1 US 20080194422 A1 US20080194422 A1 US 20080194422A1 US 10668608 A US10668608 A US 10668608A US 2008194422 A1 US2008194422 A1 US 2008194422A1
Authority
US
United States
Prior art keywords
seq
primer
primer consisting
pcr
primer set
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/106,686
Inventor
Hee-Kyun Lim
Sang-Hyo Kim
Jung-joo Hwang
Young-sun Lee
Young-A Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Samsung Electronics Co Ltd
Original Assignee
Samsung Electronics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Samsung Electronics Co Ltd filed Critical Samsung Electronics Co Ltd
Priority to US12/106,686 priority Critical patent/US20080194422A1/en
Publication of US20080194422A1 publication Critical patent/US20080194422A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • 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.
  • SARS 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 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.
  • BNI Bernhard-Nocht Institute
  • Such a narrow application site may lead to either false negative or false positive results.
  • 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.
  • 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.
  • 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 method for detecting SARS-COV which includes amplifying a nucleic acid sample obtained from an individual by PCR using the primer set.
  • a SARS-COV detection kit including the primer set.
  • 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
  • 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.
  • the present invention provides a PCR primer set selected from the group consisting of the following primer sets:
  • 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;
  • a primer set 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;
  • 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;
  • a primer set 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;
  • a primer set (e) 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;
  • a primer set 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;
  • 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;
  • a primer set (h) 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;
  • a primer set 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;
  • a primer set (u) 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;
  • 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;
  • 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.
  • 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.
  • thermal cycling e.g., 90-96° C., 72° C., and 37-55° C.
  • the PCR may be performed in a polypropylene tube, a 96-well plate, or a silicon-based micro PCR chip.
  • the 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.
  • 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.
  • the PCR may be performed using 0.2-1 ⁇ M of each primer and 0.01 pg to 1 ⁇ g of a template DNA.
  • 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.
  • 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.
  • 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;
  • a primer set (k) 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;
  • a primer set (l) 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;
  • a primer set (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;
  • a primer set (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;
  • a primer set (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;
  • 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;
  • 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;
  • 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;
  • a primer set (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;
  • a primer set (t) a primer set (GS-SARS20) including a primer having a nucleotide sequence
  • 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 ⁇ g/20 ⁇ l.
  • the RNA samples in a 25 ⁇ l 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.
  • FIG. 2 The results are shown in FIG. 2 .
  • SARS-CoV RNAs were detected.
  • 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
  • lane 6 is a PCR product (118 bp) amplified with GS-SARS04.
  • 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 ⁇ 10 3 copies.
  • the RNA samples in a 1 ⁇ l 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.
  • FIG. 3 The results are 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 .
  • PCR was performed in the same manner as in Example 3 except that 45 cycles were repeated using a 25 ⁇ l PCR solution containing 1 ng SARS-COV cDNAs+200 ng human gDNAs as templates.
  • 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
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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

    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 2.4×103 copies. 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 (10)

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.
US12/106,686 2003-12-12 2008-04-21 Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same Abandoned US20080194422A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/106,686 US20080194422A1 (en) 2003-12-12 2008-04-21 Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR1020030090559A KR100994568B1 (en) 2003-12-12 2003-12-12 A primer for detecting SARS-coronavirus, method and kit for detecting SARS-coronavirus using the same
KR2003-90559 2003-12-12
US10/997,462 US7375210B2 (en) 2003-12-12 2004-11-24 PCR primer set for detecting severe acute respiratory syndrome (SARS)-Coronavirus, method and kit for detecting SARS-Coronavirus using the same
US12/106,686 US20080194422A1 (en) 2003-12-12 2008-04-21 Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/997,462 Division US7375210B2 (en) 2003-12-12 2004-11-24 PCR primer set for detecting severe acute respiratory syndrome (SARS)-Coronavirus, method and kit for detecting SARS-Coronavirus using the same

Publications (1)

Publication Number Publication Date
US20080194422A1 true US20080194422A1 (en) 2008-08-14

Family

ID=37251954

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/997,462 Expired - Fee Related US7375210B2 (en) 2003-12-12 2004-11-24 PCR primer set for detecting severe acute respiratory syndrome (SARS)-Coronavirus, method and kit for detecting SARS-Coronavirus using the same
US12/106,686 Abandoned US20080194422A1 (en) 2003-12-12 2008-04-21 Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US10/997,462 Expired - Fee Related US7375210B2 (en) 2003-12-12 2004-11-24 PCR primer set for detecting severe acute respiratory syndrome (SARS)-Coronavirus, method and kit for detecting SARS-Coronavirus using the same

Country Status (2)

Country Link
US (2) US7375210B2 (en)
KR (1) KR100994568B1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100824414B1 (en) * 2005-11-11 2008-04-23 대한민국 - multiplex pcr method for diagnosing respiratory viruses and primers therefor
CA2629652A1 (en) * 2007-04-24 2008-10-24 Yaojiong Wu Compositions for preventing or treating skin defects and methods of use thereof
US8301359B1 (en) 2010-03-19 2012-10-30 HyCogen Power, LLC Microprocessor controlled automated mixing system, cogeneration system and adaptive/predictive control for use therewith
KR102018201B1 (en) 2019-05-01 2019-09-06 대한민국 Method and kit for detecting batcoronavirus using real-time PCR
KR102295574B1 (en) * 2020-05-14 2021-08-31 한국표준과학연구원 Human betacoronavirus universal primer sets for whole genome amplification method and diagnosis kit

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060240530A1 (en) * 2003-07-21 2006-10-26 University Of North Carolina At Chapel Hill Methods and compositions for infectious cDNA of SARS coronavirus
US7129042B2 (en) * 2003-11-03 2006-10-31 Diagnostic Hybrids, Inc. Compositions and methods for detecting severe acute respiratory syndrome coronavirus
US20060257852A1 (en) * 2003-04-10 2006-11-16 Chiron Corporation Severe acute respiratory syndrome coronavirus
US7339051B2 (en) * 2003-04-28 2008-03-04 Isis Pharmaceuticals, Inc. Compositions and methods for the treatment of severe acute respiratory syndrome (SARS)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060257852A1 (en) * 2003-04-10 2006-11-16 Chiron Corporation Severe acute respiratory syndrome coronavirus
US7339051B2 (en) * 2003-04-28 2008-03-04 Isis Pharmaceuticals, Inc. Compositions and methods for the treatment of severe acute respiratory syndrome (SARS)
US20060240530A1 (en) * 2003-07-21 2006-10-26 University Of North Carolina At Chapel Hill Methods and compositions for infectious cDNA of SARS coronavirus
US7129042B2 (en) * 2003-11-03 2006-10-31 Diagnostic Hybrids, Inc. Compositions and methods for detecting severe acute respiratory syndrome coronavirus

Also Published As

Publication number Publication date
US20080096186A1 (en) 2008-04-24
KR100994568B1 (en) 2010-11-15
KR20050058620A (en) 2005-06-17
US7375210B2 (en) 2008-05-20

Similar Documents

Publication Publication Date Title
CN111041129B (en) Primer-probe combination for detecting 6 respiratory viruses, kit and application
JP6329370B2 (en) Simultaneous diagnosis kit for diseases caused by respiratory viruses
AU675520B2 (en) Self-sustained, sequence replication system
US20050202414A1 (en) Apparatus and methods for detecting a microbe in a sample
US20080194422A1 (en) Pcr primer set detecting severe acute respiratory syndrome (sars)-coronavirus, method and kit for detecting sars-coronavirus using the same
US20170268074A1 (en) HEV Assay
US20230151444A1 (en) Pcr based diagnostic kit, compositions and methods for amplification and detection of sars-cov-2
Miao et al. Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay
US7582740B2 (en) Methods and kits for detecting SARS-associated coronavirus
CN112725539B (en) RPA/Cas12a/IF kit for respiratory syncytial virus and detection method thereof
CN110527747B (en) Kit for detecting wild strains of classical swine fever viruses
KR101100436B1 (en) A primer for detecting SARS-coronavirus, method and kit for detecting SARS-coronavirus using the same
CN113817870B (en) Primer composition for simultaneously detecting seven respiratory tract related viruses and application thereof
CN115852056A (en) Fluorescent PCR kit for respiratory virus detection and detection method
CN113481326B (en) Isothermal nucleic acid amplification reaction reagent, isothermal nucleic acid amplification method and application thereof
Callison et al. Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction
CN116064954A (en) Kit for detecting novel coronavirus and typing based on MB-RT-PCR and application thereof
JP2007514440A (en) Sensitive and specific test to detect SARS coronavirus
EP3933052A1 (en) Rapid detection kit for human pathogenic coronaviruses : betacoronavirus group b/c and sars-cov-2
Poddar Detection of type and subtypes of influenza virus by hybrid formation of FRET probe with amplified target DNA and melting temperature analysis
Diego et al. A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP assay to detect SARS-CoV-2
Werling et al. Ability to differentiate between cp and ncp BVDV by microarrays: Towards an application in clinical veterinary medicine?
EP1203826B1 (en) Oligonucleotide for detection of HIV-1 and detection method
WO2022210122A1 (en) Set of oligonucleotides for detecting plurality of types of viruses through multiplex pcr
KR102555480B1 (en) CRISPR-Cas12a-based detection of influenza virus types

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION