CN113684320A - Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof - Google Patents
Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof Download PDFInfo
- Publication number
- CN113684320A CN113684320A CN202111117248.2A CN202111117248A CN113684320A CN 113684320 A CN113684320 A CN 113684320A CN 202111117248 A CN202111117248 A CN 202111117248A CN 113684320 A CN113684320 A CN 113684320A
- Authority
- CN
- China
- Prior art keywords
- seq
- nucleotide sequences
- novel coronavirus
- sequences shown
- cov
- 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.)
- Pending
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
- C12Q1/70—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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application discloses a primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof. The primer group is designed by taking the N gene of the novel coronavirus SARS-CoV-2 as a target gene, and based on the advantages of high specificity, high sensitivity and convenience of the loop-mediated isothermal amplification technology, a set of novel coronavirus constant-temperature rapid nucleic acid amplification kit is provided, the novel coronavirus can be rapidly and accurately detected by using the kit, and the primer group has wide application prospect.
Description
Technical Field
The application relates to the technical field of nucleic acid amplification, in particular to a primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof.
Background
The novel Coronavirus belongs to the order of Nidovirales (Nidovirales), the family of Coronaviridae (Coronaviridae) and the genus Coronaviridae (Coronavir), has the characteristics of the largest class of RNA viruses discovered at present, and the genome of the Coronavirus comprises 16 non-structural protein genes (NSPs) and 4 structural protein genes (S, M, E and N proteins). The N gene and ORF1ab gene were amplified by the fluorescent quantitative PCR method.
However, since the detection by the fluorescent quantitative PCR method requires a skilled technician, a precise detection instrument and expensive and scarce reagents as conditions, rapid amplification and analysis of nucleic acid cannot be achieved.
Loop-Mediated Isothermal Amplification (LAMP) is a new gene Amplification technology developed by Notomi in Japan in 2000, and has the advantages of strong specificity, high sensitivity, isothermy, high efficiency, simple operation, low cost and the like. However, since LAMP amplification is strand displacement synthesis, the length of a target sequence is within 300bp, and amplification is difficult when the length is more than 500bp, so that amplification of long-chain DNA cannot be performed. Because the sensitivity is high, the false positive result is easy to be generated by pollution, so the strict operation is particularly noticed to prevent the nonspecific amplification and the pollution.
Disclosure of Invention
In view of the above, the present application aims to provide a method for amplifying a target sequence with a length greater than 500bp (e.g., a length of a cDNA sequence of a novel coronavirus N gene greater than 500bp) by using LAMP-PCR technology, and to reduce the problems of non-specific amplification and contamination. For this reason, the present application provides the following technical solution, which can solve at least one of these problems to some extent.
The first aspect of the embodiments of the present application provides a primer set for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, comprising a first primer pair, a second primer pair, and a third primer pair;
the first primer pair comprises nucleotide sequences shown as SEQ ID NO.31 and SEQ ID NO.32 and nucleotide sequences shown as SEQ ID NO. 31-32 obtained by substituting, deleting or adding one or more bases to the nucleotide sequences shown as SEQ ID NO. 31-32, wherein the nucleotide sequences are functionally identical or similar to the nucleotide sequences shown as SEQ ID NO. 31-32;
the second primer pair comprises nucleotide sequences shown as SEQ ID NO.33 and SEQ ID NO.34 and nucleotide sequences shown as SEQ ID NO. 33-34 obtained by substituting, deleting or adding one or more bases to the nucleotide sequences shown as SEQ ID NO. 33-34, wherein the nucleotide sequences have the same or similar functions;
the third primer pair comprises nucleotide sequences shown as SEQ ID NO.35 and SEQ ID NO.36 and nucleotide sequences shown as SEQ ID NO. 35-36 obtained by substituting, deleting or adding one or more bases in the nucleotide sequences shown as SEQ ID NO. 35-36, wherein the nucleotide sequences are functionally identical or similar to the nucleotide sequences shown as SEQ ID NO. 35-36.
In the embodiment of the application, the reverse transcription cDNA sequence of the novel coronavirus N gene is shown as SEQ ID NO.37, and the primer group targets 12-213 nt regions of the sequence shown as SEQ ID NO. 37.
The second aspect of the embodiments of the present application also provides a reaction system for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, including the primer set.
In the embodiment of the application, the reaction system also comprises Bst DNA polymerase, dNTPs, thermostable transcriptase, trehalose, (NH)4SO4Visual fluorescent dye, taurine, Tris-HCl, TrionX-100 and Mg-containing2+At least one of the reaction buffers of (1).
In the embodiment of the application, the reaction system further comprises a positive template, and the positive template is a DNA molecule with the nucleotide sequence shown in SEQ ID NO.37, or a DNA molecule which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in SEQ ID NO.37 and has the same or similar functions with the nucleotide sequence shown in SEQ ID NO. 37.
In the embodiment of the application, the reaction temperature of the reaction system is 55-62 ℃.
The third aspect of the embodiments of the present application also provides a kit for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, comprising the primer set of the first aspect and an acceptable auxiliary agent for virus detection.
The fourth aspect of the embodiments of the present application also provides a use of the primer set or the reaction system in the preparation of a reagent and/or a kit for detecting a novel coronavirus.
Compared with the prior art, the application has at least the following beneficial effects:
the primer groups are provided, the design is carried out aiming at the N gene of the novel coronavirus, and the double strand of the cDNA of the gene is divided into six specific intervals, so that the primers form two annular structures in the amplification process, isothermal amplification is realized, the effective amplification can be realized, and the primers have strong specificity and high sensitivity.
The LAMP kit for detecting the novel coronavirus combines the primer combination with the LAMP technology, can observe a detection result through naked eyes, does not need complex auxiliary equipment, is simple and easy to use, can realize semi-quantification of the detection result through a fluorescent dye method, has high detection sensitivity, and provides a convenient and quick means for diagnosis of related diseases of the novel coronavirus and research of treatment medicines.
Drawings
FIG. 1 is a schematic diagram of targeting region of primer set on SARS-CoV-2N gene sequence provided in the examples of the present application.
FIG. 2 is an electrophoresis diagram and an amplification curve of DNA products amplified at different temperatures by using different PS1 (FIG. 2A), PS2 (FIG. 2B) and PS3 (FIG. 2C) primer sets with different standards as templates provided in the examples of the present application.
FIG. 3 is an electrophoresis diagram and an amplification curve of DNA products amplified at different temperatures by using different primers of PS4 (FIG. 3D), PS5 (FIG. 3E) and PS6 (FIG. 3F) and different standards as templates according to the present application.
FIG. 4 shows the different amplifications of the PS6 primer set provided in the examples of the present applicationElectrophorograms and amplification curves of the DNA products of the templates with different concentrations under time; at different copy number concentrations (10, 10)3、105、107copies/. mu.L) as a template, and amplifying at a constant temperature of 58 ℃ for different periods of time (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 min).
FIG. 5 is an electrophoresis chart and an amplification curve of DNA products of different source templates with a PS6 primer set provided in the present application example, which is amplified for 40min at a constant temperature of 58 ℃.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Unless otherwise specified, the reagents and equipment used in the examples of the present application are all used in routine experiments, and do not limit the experimental effects of the present application.
Primer set design
SARS-CoV-2N Gene is used as a target Gene to design a primer group, N Gene NC-045512.2, Gene ID 43740575 and 1260bp ss-RNA are shown as SEQ ID NO.37, the primer group targets 12-213 nt region, 441-635 nt region, 81-296 nt region or 429-641 nt region of a sequence shown as SEQ ID NO.37, correspondingly designed primer groups are shown as table 1, and PS1-PS6 and 6 groups of primers are designed together. Wherein F3/B3 is used as a first primer pair, FIP/BIP is used as a second primer pair, and LF/LB is used as a third primer pair.
TABLE 1
The above primers were synthesized by Biotech limited of Beijing Optimalaceae, all primers DNA dry powder was dissolved in sterile RNase-free deionized water to 100. mu.M mother liquor, and 20. mu.L of the mother liquor was diluted with 80. mu.L of pure water to 20. mu.M of the use concentration.
Therefore, the efficiency and sensitivity of amplification can be improved by using the first primer pair, the second primer pair and the third primer pair, which is verified by the following embodiments.
Amplification Effect of primer set
The primer group is used for amplifying and detecting the nucleic acid of the novel coronavirus respectively with the primer group provided by the literature.
Using the primer sets provided in the present application, and a primer solution prepared at 100. mu.M with DEPC-H20, respectively, SARS-CoV-2N gene plasmid (Nanjing Kingsler Co.) was amplified using the reaction system shown in Table 2, and examples 1 to 6 were formed in this order.
TABLE 2
In table 2, the preparation method of the template is: the plasmid pcDNA3.1-N-HA carrying SARS-CoV-2N gene HAs mass concentration of 809 ng/microliter, total base number of the plasmid is 6000bp, and its relative molecular mass is calculated to be 6000 × 660-3.96 × 106. According to the formula: [ plasmid copy number ═ (plasmid mass concentration ÷ molecular weight) × 6.02 × 1023]Use of ddH on the original plasmid solution2Diluting with O to obtain 1X 108Copy number/. mu.L, 1X 107Copy number/. mu.L, 1X 106Copy number/. mu.L, 1X 105Copy number/. mu.L, 1X 104Copy number/. mu.L, 1X 103Copy number/. mu.L, 1X 102Copy number/. mu.L and 10 copy number/. mu.L of template solution.
The results show that example 1 did not amplify the fragment (FIG. 2A); example 2 (FIG. 2B), example 3 (FIG. 2C), example 4 (FIG. 3D) and example 5 (FIG. 3E) all amplified specific fragments at 55 deg.C, 58 deg.C and 60 deg.C, but did not amplify low copy number templates and were not sensitive. In contrast, example 6 amplified fragments sensitively at 55 ℃, 58 ℃ and 60 ℃ and had optimal reaction sensitivity and specificity at 58 ℃ (FIG. 3F).
Determination of reaction time and specificity of PS6
In a further embodiment, the reaction time of the PS6 primer group at different copy number concentrations is detected under the constant temperature reaction condition of 58 ℃, and the result is shown in FIG. 4, wherein the reaction time is 20min at the fastest time, the reaction time is 25min at the best, and the reaction time is kept stable for 35-45 min. Therefore, the LAMP reaction system has high sensitivity and is rapid and stable.
In a further example, LAMP-isothermal amplification was carried out using the SARS-CoV-2N gene as a positive control, by using DNA templates from different sources, including adenovirus type 3 (abbreviated as ADV-3, from the laboratory of Odonthongchow virology, Chainan university), influenza virus (H3N2-cDNA, from the laboratory of Odonthongchow virology, Chinay university), Streptococcus pneumoniae (Streptococcus pneumaniana, purchased from the center for type culture Collection of China) and Staphylococcus aureus (Straphylococcus aureus, purchased from the center for type culture Collection of China), and only the SARS-CoV-2N gene amplified a specific band, whereas DNA from other common respiratory pathogens did not amplify a band, as shown in FIG. 5. Therefore, the LAMP reaction system has high detection specificity and no cross reaction.
Nucleic acid detection kit
In this example, a kit for detecting SARS-CoV-2 nucleic acid of a novel coronavirus is prepared using the above reagents of the primer set PS1-PS6, and LMAP detection is performed on 4000copies/mL cDNA (5. mu.L for each reaction) from a throat swab of a new coronary patient using this kit, while using a 20copies standard and blank water as references, and using a multifunctional microplate reader to detect the absorbance OD650 of the product at 650 nm. The LAMP amplification reaction system is shown in Table 3.
TABLE 3 reaction System
The results of 8 pharyngeal swab samples, 1 NC sample, and 1 standard (positive control) were measured using the above reaction system and are shown in Table 4.
As can be seen from Table 4, the samples of comparative examples 2-5 and example 1 can be correctly detected, the amplification time of the PS6 primer set in the experiment of example 1 is earlier than that of comparative examples 2-5, and the amplification efficiency is higher than that of comparative examples 2-5. Therefore, the kit provided by the embodiment of the application can realize the detection of the throat swab sample containing 20copies SARS-CoV-2 only in 20min, and has high detection sensitivity and efficiency.
TABLE 4 OD650
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Sequence listing
<110> Foshan pathogenic microorganism research institute
<120> primer set for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and use thereof
<160> 37
<170> SIPOSequenceListing 1.0
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 3
cacccgcaat cctgctaac 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 5
tgctcccttc tgcgtagaag caatgctgca atcgtgctac a 41
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 6
ggcggcagtc aagcctcttc cctactgctg cctggagtt 39
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 7
gcaatgttgt tccttgagga agt 23
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 8
ttcctcatca cgtagtcgca acagt 25
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 10
ccgtcaccac cacgaatt 18
<210> 11
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 11
agcggtgaac caagacgcag ggcgcgatca aaacaacg 38
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 12
aattccctcg aggacaaggc gagctcttcg gtagtagcca a 41
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 13
ttattgggta aaccttgggg c 21
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 14
tccaattaac accaatagca gtcca 25
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 15
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 16
ccgtcaccac cacgaatt 18
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 17
agcggtgaac caagacgcag ggcgcgatca aaacaacg 38
<210> 18
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 18
aattccctcg aggacaaggc gagctcttcg gtagtagcca a 41
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 19
attattgggt aaaccttggg gc 22
<210> 20
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 20
ccaattaaca ccaatagcag tcca 24
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 21
agatcacatt ggcacccg 18
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 22
<210> 23
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 23
tgctcccttc tgcgtagaag ccaatgctgc aatcgtgcta c 41
<210> 24
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 24
ggcggcagtc aagcctcttc cctactgctg cctggagtt 39
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 25
gcaatgttgt tccttgagga agtt 24
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 26
gttcctcatc acgtagtcgc aaca 24
<210> 27
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 27
agatcacatt ggcacccg 18
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
<210> 29
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 29
tgctcccttc tgcgtagaag ccaatgctgc aatcgtgcta c 41
<210> 38
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 38
ggcggcagtc aagcctcttc cctactgctg cctggagtt 39
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 30
ggcaatgttg ttccttgagg aagtt 25
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 31
gttcctcatc acgtagtcgc aaca 24
<210> 32
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 32
tggaccccaa aatcagcg 18
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 33
gccttgtcct cgagggaat 19
<210> 34
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 34
ccactgcgtt ctccattctg gtaaatgcac cccgcattac g 41
<210> 35
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 35
cgcgatcaaa acaacgtcgg cccttgccat gttgagtgag a 41
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 36
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 37
tacccaataa tactgcgtct tggt 24
<210> 43
<211> 1260
<212> DNA
<213> Artificial Sequence
<400> 43
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260
Claims (8)
1. A primer set for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, comprising a first primer pair, a second primer pair and a third primer pair;
the first primer pair comprises nucleotide sequences shown as SEQ ID NO.31 and SEQ ID NO.32 and nucleotide sequences shown as SEQ ID NO. 31-32 obtained by substituting, deleting or adding one or more bases to the nucleotide sequences shown as SEQ ID NO. 31-32, wherein the nucleotide sequences are functionally identical or similar to the nucleotide sequences shown as SEQ ID NO. 31-32;
the second primer pair comprises nucleotide sequences shown as SEQ ID NO.33 and SEQ ID NO.34 and nucleotide sequences shown as SEQ ID NO. 33-34 obtained by substituting, deleting or adding one or more bases to the nucleotide sequences shown as SEQ ID NO. 33-34, wherein the nucleotide sequences have the same or similar functions;
the third primer pair comprises nucleotide sequences shown as SEQ ID NO.35 and SEQ ID NO.36 and nucleotide sequences shown as SEQ ID NO. 35-36 obtained by substituting, deleting or adding one or more bases in the nucleotide sequences shown as SEQ ID NO. 35-36, wherein the nucleotide sequences are functionally identical or similar to the nucleotide sequences shown as SEQ ID NO. 35-36.
2. The primer set according to claim 1, wherein the reverse transcription cDNA sequence of the N gene of the novel coronavirus is shown as SEQ ID NO.37, and the primer set targets 12-213 nt region of the sequence shown as SEQ ID NO. 37.
3. A reaction system for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, comprising the primer set of claim 1 or 2.
4. The reaction system of claim 3, further comprising Bsm DNA polymerase, dNTPs, thermostable transcriptase, trehalose, (NH)4SO4Visual fluorescent dye, taurine, Tris-HCl, TrionX-100 and Mg-containing2+At least one of the reaction buffers of (1).
5. The reaction system of claim 4, further comprising a positive template, wherein the positive template is a DNA molecule having the nucleotide sequence shown in SEQ ID No.37, or a DNA molecule which is obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID No.37 and has the same or similar function as the nucleotide sequence shown in SEQ ID No. 37.
6. The reaction system according to any one of claims 3 to 5, wherein the reaction temperature is 55 to 62 ℃.
7. A kit for amplifying or detecting a novel coronavirus SARS-CoV-2 nucleic acid, comprising the primer set of claim 1 or 2 and acceptable auxiliary agents for virus detection.
8. Use of the primer set of claim 1 or 2, or the reaction system of any one of claims 3 to 6, for the preparation of a reagent and/or a kit for the detection of a novel coronavirus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111117248.2A CN113684320A (en) | 2021-09-23 | 2021-09-23 | Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111117248.2A CN113684320A (en) | 2021-09-23 | 2021-09-23 | Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113684320A true CN113684320A (en) | 2021-11-23 |
Family
ID=78586957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111117248.2A Pending CN113684320A (en) | 2021-09-23 | 2021-09-23 | Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113684320A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774589A (en) * | 2022-05-21 | 2022-07-22 | 中国科学院青岛生物能源与过程研究所 | Primer group, reagent and kit for detecting SARS-CoV-2 and mutant strain thereof and application |
| CN116590387A (en) * | 2023-07-06 | 2023-08-15 | 深圳大学 | CRISPR (clustered regularly interspaced short palindromic repeats) system-based ssDNA detection method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982944A (en) * | 2020-03-03 | 2020-04-10 | 中国农业科学院北京畜牧兽医研究所 | Novel visualized constant-temperature rapid detection kit for coronavirus |
| CN112626266A (en) * | 2020-10-27 | 2021-04-09 | 山西高等创新研究院 | Novel detection primer group for coronavirus SARS-CoV-2 and application thereof |
-
2021
- 2021-09-23 CN CN202111117248.2A patent/CN113684320A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982944A (en) * | 2020-03-03 | 2020-04-10 | 中国农业科学院北京畜牧兽医研究所 | Novel visualized constant-temperature rapid detection kit for coronavirus |
| CN112626266A (en) * | 2020-10-27 | 2021-04-09 | 山西高等创新研究院 | Novel detection primer group for coronavirus SARS-CoV-2 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| WEI E. HUANG等: "RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2", MICROBIAL BIOTECHNOLOGY, vol. 13, no. 4, pages 951 - 955 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774589A (en) * | 2022-05-21 | 2022-07-22 | 中国科学院青岛生物能源与过程研究所 | Primer group, reagent and kit for detecting SARS-CoV-2 and mutant strain thereof and application |
| CN116590387A (en) * | 2023-07-06 | 2023-08-15 | 深圳大学 | CRISPR (clustered regularly interspaced short palindromic repeats) system-based ssDNA detection method and application |
| CN116590387B (en) * | 2023-07-06 | 2023-12-08 | 深圳大学 | CRISPR (clustered regularly interspaced short palindromic repeats) system-based ssDNA detection method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP4202064A1 (en) | Kit and method for isothermal rapid detection of sars-cov-2 virus nucleic acid | |
| CN111500771B (en) | Primer group and kit for detecting novel coronavirus SARS-CoV-2 | |
| WO2023109032A1 (en) | Multiple nucleic acid detection system, and preparation method therefor and use thereof | |
| CN110408725B (en) | Kit for multiple detection of respiratory pathogens | |
| CN113046475B (en) | Primer composition and kit for rapidly detecting mutant novel coronavirus | |
| CN113684320A (en) | Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof | |
| CN110273026B (en) | Multiple detection kit and detection method for respiratory tract infection | |
| JP7313537B2 (en) | Method for detecting coronavirus (SARS-CoV-2) | |
| CN112877410B (en) | Optimized nucleic acid detection system based on CRISPR (clustered regularly interspaced short palindromic repeats) mediation and detection method thereof | |
| CN110791578A (en) | CRISPR (clustered regularly interspaced short palindromic repeats) detection primer group for bordetella pertussis and application of CRISPR detection primer group | |
| CN113151590A (en) | Novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof | |
| JP2019013248A (en) | Compositions and methods for detecting hev nucleic acid | |
| US20240076712A1 (en) | Compositions and methods for instant nucleic acid detection | |
| Li et al. | Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection | |
| CN111719015A (en) | Human immunodeficiency virus HIV-1 detection kit | |
| CN116287475A (en) | Nucleic acid sequence combination, kit and application of brassica yellow virus RT-LAMP-CRISPR isothermal detection | |
| CN116555495A (en) | Primer probe combination, kit and method for identifying and detecting African swine fever I177L gene deletion strain | |
| CN110724763A (en) | Fluorescent quantitative PCR detection method for human adenovirus and bocavirus and application thereof | |
| CN115838834A (en) | Human rhinovirus type A and type C detection system based on RT-RAA and CRISPR/Cas12a | |
| Taggart et al. | Use of heat labile UNG in an RT-PCR assay for enterovirus detection | |
| CN116103437A (en) | Kit and primer combination and method for LAMP detection of influenza A virus, influenza B virus and respiratory syncytial virus | |
| CN115029345A (en) | Nucleic acid detection kit based on CRISPR and application thereof | |
| JP2022105717A (en) | Compositions, methods and kits to detect adenovirus, metapneumovirus and/or rhinovirus nucleic acids | |
| CN113151585A (en) | Primer composition and kit for rapidly detecting mutation of novel coronavirus D614G | |
| CN116103438A (en) | Kit for LAMP detection of respiratory viruses and primer composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |