CN111424119B - High-flux detection primer and kit for SARS-CoV-2 virus - Google Patents
High-flux detection primer and kit for SARS-CoV-2 virus Download PDFInfo
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- CN111424119B CN111424119B CN202010489954.9A CN202010489954A CN111424119B CN 111424119 B CN111424119 B CN 111424119B CN 202010489954 A CN202010489954 A CN 202010489954A CN 111424119 B CN111424119 B CN 111424119B
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- 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
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- 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
- C12Q1/6869—Methods for sequencing
Abstract
The invention relates to the technical field of detection reagents, in particular to a high-throughput detection primer and a kit for SARS-CoV-2 virus. The primer provided by the invention is used for amplifying specific regions in SARS-CoV-2 virus, and the fragments are used as detection markers of SARS-CoV-2 virus, so that high-throughput detection of SARS-CoV-2 virus can be realized, and the primer has high sensitivity, good specificity and high accuracy.
Description
Technical Field
The invention relates to the technical field of detection reagents, in particular to a high-throughput detection primer and a kit for SARS-CoV-2 virus.
Background
2019 the novel coronavirus (SARS-CoV-2) is a new strain of coronavirus found in human in 2019. The symptoms of the virus are fever, hypodynamia, dry cough and gradual dyspnea, and severe patients show acute respiratory distress syndrome, septic shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct. The virus has infectivity in latent period, and no specific treatment method for the disease exists at present.
In order to control the development of the epidemic as soon as possible, it is important to test all patients likely to be infected with SARS-CoV-2 virus within the first time. At present, the main method for detecting SARS-CoV-2 virus is real-time fluorescent quantitative PCR technology, which has the characteristics of strong specificity and high sensitivity, but because the method has higher requirements on operators and the used detection instrument limits the number of samples to be detected at one time, the flux of the samples to be detected at one time is lower, and the detection requirements of a large number of samples cannot be met, the reagent capable of detecting SARS-CoV-2 virus at high flux is urgently needed at present.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a primer and a kit for high-throughput detection of SARS-CoV-2 virus, wherein the primer is suitable for a nucleic acid detection method of high-throughput sequencing.
The invention provides application of at least two of 7 nucleic acid fragments shown in SEQ ID NO. 1-7 as markers in preparation of a SARS-CoV-2 virus detection reagent.
The invention also provides a high-throughput detection primer group of SARS-CoV-2 virus, the upstream primer of the primer group consists of an upstream general sequence and an upstream recognition sequence which are connected in sequence;
the downstream primer consists of a downstream general sequence 1, a tag sequence, a downstream general sequence 2 and a downstream identification sequence which are connected in sequence;
wherein, the nucleotide sequence of the upstream universal sequence is shown as SEQ ID NO. 8;
the nucleotide sequence 1 of the downstream universal sequence is shown as SEQ ID NO. 9;
the nucleotide sequence 2 of the downstream universal sequence is shown as SEQ ID NO. 10;
the upstream recognition sequence is selected from any one of 7 sequences shown in SEQ ID NO. 11-17;
the downstream recognition sequence is selected from any one of 7 sequences shown in SEQ ID NO 18-24.
The primer group also comprises internal reference primers shown as SEQ ID NO. 25-26.
In some embodiments, the primer set comprises 7 pairs of specific primers and reference primers shown as SEQ ID NO. 25-26;
the primer pair 1 comprises:
an upstream primer 1:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTGATGGTGGTGTCACTCGTG-3’
a downstream primer 1:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGGCACGACAAAACCCACTTC-3'
The primer pair 2 comprises:
an upstream primer 2:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAACTGCACCTCATGGTC-3’
a downstream primer 2:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGACTTTAGATCGGCGCCGTA-3'
The primer pair 3 comprises:
an upstream primer 3:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC TGTCGTTGACAGGACACGAG-3’
a downstream primer 3:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCAGTCTCCAAAGCCACGTACG-3'
The primer pair 4 comprises:
an upstream primer 4:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC CGGATGGCTTATTGTTGGCG-3’
a downstream primer 4:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGGCATTTCCAGCAAAGCCAA-3'
The primer pair 5 comprises:
an upstream primer 5:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC GCCGCTGTTGATGCACTATG-3’
a downstream primer 5:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCGTCTCAGGCAATGCAT-3'
The primer pair 6 comprises:
an upstream primer 6:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC TCTTCTCAACGTGCCACTCC-3’
a downstream primer 6:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGGCAGGTCCTTGATGTCACA-3'
The primer pair 7 includes:
an upstream primer 7:
5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC ACACGTAACCCTGCTTGGAG-3’
a downstream primer 7:
5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCTGAGGTGTGTAGGTGCCTG-3'.
In the primer group, the tag sequence is random, and the length of the tag sequence is 8 bp; each detection system corresponds to a tag sequence.
Each sample to be detected corresponds to a label sequence, and each detection system at least comprises two pairs of detection primers and one pair of internal reference primers. The tag sequences of all primers in each detection system are identical.
The tag sequence is used in sequencing to distinguish between samples to be tested. The primer group provided by the invention is suitable for high-throughput detection of SARS-CoV-2 virus, the detection of the primer group depends on an Illumina sequencer, and a sequencing strategy uses single-ended 150p sequencing. Based on the application of a high-throughput platform, the primer group can be used for simultaneously detecting a plurality of samples, and the maximum detectable sample amount in each detection can reach 1000 cases. Thus, the tag sequence is randomly selected from:
the invention also provides a high-throughput detection kit for SARS-CoV-2 virus, which comprises the primer group of the invention.
The high-throughput detection kit also comprises PCR buffer, dNTP mix and Taq DNA Polymerase.
In the invention, the PCR buffer (with the pH value of 7.5-8.5) comprises 30mM tris-HCl, 50mM KCl, 2mM MgCl2 and 10mM ammonium sulfate.
The composition of the dNTP mix is: 100mM dATP, 100mM dCTP, 100mM dGTP and 100mM dTTP.
The invention also provides a SARS-CoV-2 virus detection method of non-diagnosis purpose, which comprises amplifying sample cDNA by the primer group of the invention, judging whether the sample contains SARS-CoV-2 virus according to the sequencing result of the amplification product;
the judging method comprises the following steps: if the reads number of at least two markers in the markers detected by one sample is more than 10, judging the markers to be positive results, otherwise, judging the markers to be negative results; the marker is the marker of the invention.
In the invention, before sequencing the amplification product, the method also comprises a purification step, wherein the amplification product is purified by using AMPure XP magnetic beads.
In the present invention, the amplification system comprises:
the primer mixture is prepared by mixing the primer group of the invention with equal amount (the total concentration is 20 mu M).
In the present invention, the procedure of amplification is: 4min at 96 ℃; 20s at 96 ℃, 30s at 59 ℃, 40s at 72 ℃ and 16 cycles; 10min at 72 ℃.
The primer provided by the invention is used for amplifying specific regions in SARS-CoV-2 virus, and the fragments are used as detection markers of SARS-CoV-2 virus, so that high-throughput detection of SARS-CoV-2 virus can be realized, and the primer has high sensitivity, good specificity and high accuracy.
Detailed Description
The invention provides a high-flux detection primer and a kit for SARS-CoV-2 virus, and the technical personnel in the field can use the content for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The corresponding genes and positions of the markers adopted by the invention in SARS-CoV-2 virus are shown in Table 1:
TABLE 1 marker sequences
The invention is further illustrated by the following examples:
example 1
1. 200 samples of the sample types of ordinary human sputum and throat swab and 10 artificial RNA-mimetic mixtures (the sequence of which coincides with SARS-CoV-2 virus sequence) were taken as test samples.
2. Nucleic acid extraction: 200 samples of sputum and pharyngeal swab nucleic acids were extracted using the QIAamp Viral RNA Mini Kit, and the specific procedures were performed according to the instruction manual.
3. The extracted nucleic acid RNA and the artificial RNA are reversely transcribed into cDNA by using a Thermo Fisher reverse transcription kit, and the specific operation is carried out according to the instruction program.
4. And (3) performing multiplex PCR amplification on each sample corresponding to a tag primer mix by using the cDNA obtained in the step (3) as a template. The reaction system is as follows: mu.L of primer mixture mix, 12 mu.L of PCR buffer (pH 7.5-8.5), 0.8 mu.L of dNTP mix, 0.8 mu.L of Taq DNA Polymerase (2.5U/. mu.L), 5 mu.L of sample to be detected, and 4.4 mu.L of nucleic acid-free water to a total reaction volume of 25 mu.L. The primer sequences are shown in Table 2:
TABLE 2 primer sequences
5. The reaction procedure was as follows: 4min at 96 ℃; 20s at 96 ℃, 30s at 59 ℃, 40s at 72 ℃ and 16 cycles; 10min at 72 ℃.
6. The product of the above reaction was purified using AMPure XP magnetic beads.
7. And carrying out quantitative and quality detection on the purified sample.
8. All samples were pooled and sequenced on an Illumina sequencer using a single-ended 150p sequencing strategy.
9. And (5) analyzing the letter and judging the result. Filtering data which do not meet the quality requirement, comparing the sample use tag sequence and the sequencing sequence with an analysis use database, and analyzing 200 samples of sputum and throat swab which are all negative, 10 samples of artificially synthesized RNA which are positive are consistent with the expected result, wherein the accuracy is 100%.
Example 2
The experimental method comprises the following steps: the method provided in example 1 was applied to the detection of SARS-CoV-2 virus in environmental water, soil or non-biological surfaces using 5 samples of tap water spiked with positive plasmids and 10 samples of tap water.
1. And concentrating and enriching the viruses in the water body sample by adopting an anion membrane adsorption-ultrasonic oscillation method.
2. Nucleic acid extraction: tap water sample nucleic acid was extracted using the QIAamp Viral RNA Mini Kit, and the detailed procedures were performed with reference to the instruction manual.
3. The extracted nucleic acid RNA is reversely transcribed into cDNA by using a Thermo Fisher reverse transcription kit, and the specific operation is carried out according to the instruction program.
4. And performing multiplex PCR amplification on each sample corresponding to one tag primer mix by using the obtained cDNA as a template. The reaction system is as follows: primer mix 2 μ L, PCR buffer (pH 7.5-8.5)12 μ L, dNTP mix 0.8 μ L, Taq DNA Polymerase (2.5U/μ L)0.8 μ L, template to be detected 5 μ L, supplement nucleic acid free 4.4 μ L to total reaction volume 25 μ L
5. The reaction procedure was as follows: 4min at 96 ℃; 20s at 96 ℃, 30s at 59 ℃, 40s at 72 ℃ and 16 cycles; 10min at 72 ℃.
6. The product of the above reaction was purified using AMPure XP magnetic beads.
7. And carrying out quantitative and quality detection on the purified sample.
8. All samples were pooled and sequenced on an Illumina sequencer using a single-ended 150p sequencing strategy.
9. And (5) analyzing the letter and judging the result. And filtering data which do not meet the quality requirement, comparing the sample use label sequence and the sequencing sequence with an analysis use database, and analyzing to obtain 10 parts of tap water samples which are all negative and 8 parts of tap water samples doped with positive plasmids which are positive. The results were consistent with expected results with 100% accuracy.
Example 3
Purpose of the experiment: detecting the accuracy of the detection reagent
The experimental method comprises the following steps: the sequencing library obtained by the method provided in example 1 and a metagenome whole gene sequencing library constructed by using the same genome as a template are subjected to high-throughput sequencing by using an Illumina NextSeq550 sequencer respectively, and sequencing data and detection results of the two libraries are compared. Samples 1, 2, 3 used were known positive plasmids.
TABLE 3 test results of the accuracy test
Example 4
Purpose of the experiment: detecting the specificity of the detection reagent
The experimental method comprises the following steps: the sequencing library obtained by the method provided in example 1 was subjected to high throughput sequencing using Illumina NextSeq550 sequencer, and the detection results were analyzed. The samples 4, 5, 6, 7, 8, 9 used were known negative samples.
TABLE 4 detection results of specificity experiments
Example 5
Purpose of the experiment: sensitivity of detecting the detection reagent
The experimental method comprises the following steps: the sequencing library obtained by the method provided in example 1 was subjected to high throughput sequencing using illuminainnextseq 550 sequencer, and the results of the assay were analyzed. The samples used were marker plasmids containing each concentration gradient.
TABLE 5 sensitivity test results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Mitsubishi medical science and technology (Beijing) Co., Ltd
<120> SARS-CoV-2 virus high flux detection primer and reagent kit
<130> MP2004120
<160> 26
<170> SIPOSequenceListing 1.0
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<212> DNA
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tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 60
tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 120
attgattgct gcagtcataa caagagaagt gggttttgtc gtgcc 165
<210> 2
<211> 198
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tcgaactgca cctcatggtc atgttatggt tgagctggta gcagaactcg aaggcattca 60
gtacggtcgt agtggtgaga cacttggtgt ccttgtccct catgtgggcg aaataccagt 120
ggcttaccgc aaggttcttc ttcgtaagaa cggtaataaa ggagctggtg gccatagtta 180
cggcgccgat ctaaagtc 198
<210> 3
<211> 218
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tgtcgttgac aggacacgag taactcgtct atcttctgca ggctgcttac ggtttcgtcc 60
gtgttgcagc cgatcatcag cacatctagg tttcgtccgg gtgtgaccga aaggtaagat 120
ggagagcctt gtccctggtt tcaacgagaa aacacacgtc caactcagtt tgcctgtttt 180
acaggttcgc gacgtgctcg tacgtggctt tggagact 218
<210> 4
<211> 272
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cggatggctt attgttggcg ttgcacttct tgctgttttt cagagcgctt ccaaaatcat 60
aaccctcaaa aagagatggc aactagcact ctccaagggt gttcactttg tttgcaactt 120
gctgttgttg tttgtaacag tttactcaca ccttttgctc gttgctgctg gccttgaagc 180
cccttttctc tatctttatg ctttagtcta cttcttgcag agtataaact ttgtaagaat 240
aataatgagg ctttggcttt gctggaaatg cc 272
<210> 5
<211> 168
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gccgctgttg atgcactatg tgagaaggca ttaaaatatt tgcctataga taaatgtagt 60
agaattatac ctgcacgtgc tcgtgtagag tgttttgata aattcaaagt gaattcaaca 120
ttagaacagt atgtcttttg tactgtaaat gcattgcctg agacgaca 168
<210> 6
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tcttctcaac gtgccactcc atggcactat tctgaccaga ccgcttctag aaagtgaact 60
cgtaatcgga gctgtgatcc ttcgtggaca tcttcgtatt gctggacacc atctaggacg 120
ctgtgacatc aaggacctgc c 141
<210> 7
<211> 386
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acacgtaacc ctgcttggag aaaagctgtc tttatttcac cttataattc acagaatgct 60
gtagcctcaa agattttggg actaccaact caaactgttg attcatcaca gggctcagaa 120
tatgactatg tcatattcac tcaaaccact gaaacagctc actcttgtaa tgtaaacaga 180
tttaatgttg ctattaccag agcaaaagta ggcatacttt gcataatgtc tgatagagac 240
ctttatgaca agttgcaatt tacaagtctt gaaattccac gtaggaatgt ggcaacttta 300
caagctgaaa atgtaacagg actctttaaa gattgtagta aggtaatcac tgggttacat 360
cctacacagg cacctacaca cctcag 386
<210> 8
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aatgatacgg cgaccaccga gatctacaca cactctttcc ctacacgacg ctcttccgat 60
c 61
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caagcagaag acggcatacg agat 24
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<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtgactggag ttcagacgtg tgctcttccg atc 33
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgatggtggt gtcactcgtg 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcgaactgca cctcatggtc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tgtcgttgac aggacacgag 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cggatggctt attgttggcg 20
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gccgctgttg atgcactatg 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tcttctcaac gtgccactcc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
acacgtaacc ctgcttggag 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggcacgacaa aacccacttc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gactttagat cggcgccgta 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
agtctccaaa gccacgtacg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggcatttcca gcaaagccaa 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tgtcgtctca ggcaatgcat 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ggcaggtcct tgatgtcaca 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctgaggtgtg taggtgcctg 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gatttggtcg tattgggcgc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aaatgagccc cagccttctc 20
Claims (8)
- A primer group for high-flux detection of SARS-CoV-2 virus, which is characterized by consisting of 7 pairs of specific primers and an internal reference primer;the primer pair 1 comprises:an upstream primer 1:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTGATGGTGGTGTCACTCGTG -3’a downstream primer 1:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCGGCACGACAAAACCCACTTC-3'The primer pair 2 comprises:an upstream primer 2:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAACTGCACCTCATGGTC -3’a downstream primer 2:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCGACTTTAGATCGGCGCCGTA-3'The primer pair 3 comprises:an upstream primer 3:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC TGTCGTTGACAGGACACGAG -3’a downstream primer 3:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCAGTCTCCAAAGCCACGTACG-3'The primer pair 4 comprises:an upstream primer 4:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC CGGATGGCTTATTGTTGGCG -3’a downstream primer 4:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCGGCATTTCCAGCAAAGCCAA-3'The primer pair 5 comprises:an upstream primer 5:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC GCCGCTGTTGATGCACTATG -3’a downstream primer 5:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCTGTCGTCTCAGGCAATGCAT-3'The primer pair 6 comprises:an upstream primer 6:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC TCTTCTCAACGTGCCACTCC -3’a downstream primer 6:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTT CAGACGTGTGCTCTTCCGATCGGCAGGTCCTTGATGTCACA-3'The primer pair 7 includes:an upstream primer 7:5’-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATC ACACGTAACCCTGCTTGGAG -3’a downstream primer 7:5 '-CAAGCAGAAGACGGCATACGAGAT [ tag sequence ] GTGACTGGAGTT CAGACGTGTGCTCTTCCGATCCTGAGGTGTGTAGGTGCCTG-3'.
- 2. The primer group of claim 1, wherein the sequence of the internal reference primer is shown in SEQ ID NO 25-26.
- 3. The primer set of claim 1, wherein the tag sequence is random and has a length of 8 bp; each detection system corresponds to a tag sequence.
- A high-throughput detection kit for SARS-CoV-2 virus, comprising the primer set according to any one of claims 1 to 3.
- 5. The high-throughput assay kit of claim 4, further comprising PCR buffer, dNTP mix and Taq DNA Polymerase.
- 6. A SARS-CoV-2 virus detection method for non-diagnostic purposes, which comprises amplifying a sample cDNA using the primer set according to any one of claims 1 to 3, and determining whether or not SARS-CoV-2 virus is contained in the sample based on the sequencing result of the amplified product;the judging method comprises the following steps: if the reads number of at least two markers in 7 markers detected by one sample is more than 10, judging the result as a positive result, otherwise, judging the result as a negative result;the nucleic acid sequences of the 7 markers are shown in SEQ ID NO 1-7.
- 7. The detection method according to claim 6, wherein the amplification system comprises:the primer mix was 2. mu.L total;PCR buffer 12μL；dNTP mix 0.8μL；Taq DNA Polymerase(2.5U/μL) 0.8μL；5-9.4 mu L of cDNA of a sample to be detected;make up to 25. mu.L without nucleic acid.
- 8. The detection method according to claim 6 or 7, wherein the amplification procedure is: 4min at 96 ℃; 20s at 96 ℃, 30s at 59 ℃, 40s at 72 ℃ and 16 cycles; 10min at 72 ℃.
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| CN111424119B true CN111424119B (en) | 2022-04-22 |
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