CN111500781A - Amplification primer group for detecting SARS-CoV-2 by mNGS and application thereof - Google Patents
Amplification primer group for detecting SARS-CoV-2 by mNGS and application thereof Download PDFInfo
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Abstract
The invention relates to an amplification primer group for detecting SARS-CoV-2 by using mNGS and an application thereof, belonging to the technical field of gene detection. The amplification primer set comprises: random primer and SARS-CoV-2 specific primer, the random primer is 5-7bp oligonucleotide, the SARS-CoV-2 specific primer is 10-20bp oligonucleotide designed for SARS-CoV-2 sequence. The amplification primer group is used for carrying out specific amplification on the novel coronavirus before the database construction in a database construction mode, carrying out high-coverage targeted enrichment and database construction, and improving the coverage of pathogen detection, identification of pathogen genome mutation, sensitivity of pathogen detection and diagnostic capability of novel coronavirus infection.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to an amplification primer group for detecting SARS-CoV-2 by using mNGS and application thereof.
Background
The metagenome (mNGS) technology has gradually become a widely adopted technology in many aspects of discovery and transformation research, has important application in pathogen diagnosis and analysis of urgent and difficult severe infectious diseases, and can perform unbiased sequencing on all nucleic acids in a sample, including nucleic acids of human sources and microorganisms.
The novel coronavirus, named 2019-nCoV or SARS-CoV-2, can be transmitted by spray and contact, and is a pathogen of easily transmitted diseases. The disease infected with SARS-CoV-2 is called COVID-19 pneumonia, and the symptoms are fever, dry cough, hypodynamia, etc. similar to common pneumonia, which leads to difficult identification by clinicians, and accurate identification of whether SARS-CoV-2 infection plays an important role in clinical diagnosis and treatment as well as public health safety. The new virus genome-wide sequence characteristics are the basis of the infection, transmission and pathogenic mechanism of the virus, and are the cornerstone of scientists for developing virus vaccines and medicines. The monitoring of new pathogen genome sequence mutation is an important task of disease prevention and control center.
At present, the conventional nucleic acid or immunochromatographic detection methods are limited by sensitivity, cannot effectively detect low-load viruses, and need a plurality of methods for combined detection and diagnosis. The conventional nucleic acid targeted detection method cannot identify the whole genome and mutation of the virus. The metagenome technology has unique advantages in the diagnosis of unknown pathogen infection or the confirmation of new pathogen, and makes a decisive contribution to the discovery and identification of the novel coronavirus. Because the metagenome technology is unbiased and is easily influenced by factors such as hosts, pathogen load and the like, false positive results can also appear in the detection of low-load samples. The sequencing of the viral genome needs to be carried out by virus separation and culture, which wastes time and labor and can also generate mutation in the culture process; or deep sequencing of high loads of sample nucleic acids is costly.
Disclosure of Invention
Based on the above, there is a need to provide an amplification primer set for detecting SARS-CoV-2 by using ngs and an application thereof, which can perform high coverage targeted enrichment and library construction on the novel coronavirus by adopting the primer set in a library construction manner, thereby improving the coverage of pathogen detection, identification of pathogen genome mutation, sensitivity of pathogen detection and diagnostic capability of novel coronavirus infection.
An amplification primer set for detecting SARS-CoV-2 by using mNGS comprises: random primer and SARS-CoV-2 specific primer, the random primer is 5-7bp oligonucleotide, the SARS-CoV-2 specific primer is 10-20bp oligonucleotide designed for SARS-CoV-2 sequence.
The amplification primer group is used for carrying out specific amplification on the novel coronavirus before the database construction in a database construction mode, carrying out high-coverage targeted enrichment and database construction, and improving the coverage of pathogen detection, identification of pathogen genome mutation, sensitivity of pathogen detection and diagnostic capability of novel coronavirus infection.
In one embodiment, the random primer is a 6bp oligonucleotide and the SARS-CoV-2 specific primer is a 15bp oligonucleotide designed for SARS-CoV-2 sequence.
In one embodiment, the SARS-CoV-2 specific primer is selected from at least one of the following primers:
in one embodiment, the SARS-CoV-2 specific primer is SEQ ID NO:1 to SEQ ID NO: 32 in a primer set.
In one embodiment, the working concentration of the SARS-CoV-2 specific primers is 4. + -. 0.5pM, and the amount of each primer in the specific primers is the same.
The invention also discloses the application of the amplification primer group for detecting SARS-CoV-2 by using the mNGS in the preparation of a reagent and/or equipment for detecting SARS-CoV-2.
It is understood that the product may be a kit or an integrated detection device.
The invention also discloses a reagent kit for detecting SARS-CoV-2, which comprises the amplification primer group for detecting SARS-CoV-2 by using mNGS.
In one embodiment, the kit is used to detect a nasal swab, pharyngeal swab, sputum, or alveolar lavage sample.
The invention also discloses a method for detecting SARS-CoV-2 for scientific research, which comprises the following steps:
s1: taking a biological sample, and extracting RNA to obtain an RNA sample;
s2: removing DNA in the RNA sample by using a DNA removal system to obtain an RNA pure product;
s3: using the pure RNA as a template, using the amplification primer group, and under the reverse transcription condition, adopting an RT-PCR technology to carry out targeted enrichment on SARS-CoV-2 genome, wherein each primer starts to extend from the 3' end to synthesize first strand cDNA;
s4: after the first-strand synthesis reaction is finished, adding a second-strand synthetase and a second-strand synthesis buffer solution to synthesize double-strand cDNA;
s5: using double-stranded cDNA as a template, building a transposase library, and performing PCR amplification on the library;
s6: purifying the amplified library, and sequencing by using a sequencing platform;
s7: removing the detected host sequence by bioinformatics analysis method to obtain SARS-CoV-2 genome information.
In one embodiment, in step S2, the DNA removal system includes rnase inhibitor, dnase buffer and nuclease-free water; removing DNA and then purifying magnetic beads;
in the step S4, after the reaction of synthesizing the double-stranded cDNA is completed, magnetic bead purification and Qubit quantification are performed;
in step S5, 1ng of the purified and quantified double-stranded cDNA is used as a template, and the transposase library construction method specifically comprises: fragmenting and adding a linker by adopting a Norzan TN501 transposase system, adding a fragmentation reaction termination solution after the fragmentation is finished, and then adding a labeled primer for carrying out PCR amplification of the library.
Compared with the prior art, the invention has the following beneficial effects:
the amplification primer group for detecting SARS-CoV-2 by using mNGS is characterized by that it utilizes the mode of establishing bank to make specific amplification before the bank is established, and makes high coverage target enrichment and bank-establishment so as to raise the coverage of pathogen detection, identification of pathogen genome mutation, sensitivity of pathogen detection and diagnosis capacity of new type coronavirus infection.
The primer is used as SARS-CoV-2 detecting reagent, and can raise the detection sensitivity greatly and reduce the false negative and false positive problems in conventional method.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
An amplification primer for detecting novel coronavirus (SARS-CoV-2) and a design scheme.
The invention detects the novel coronavirus SARS-CoV-2 in a targeted way, combines the advantages of the specific primer target reverse transcription and the pathogen macro gene group to carry out unbiased sequencing on all molecules in a sample, and improves the detection performance of the pathogen nucleic acid to the maximum extent. The specific primer design method comprises the following steps:
firstly, obtaining a genome sequence.
The 2019 novel coronavirus reference genome was downloaded from the NCBI website under accession number GCF _009858895.2, and the human reference genome under accession number GCF _ 000001405.39.
Secondly, generating 2019 novel coronavirus genome short sequences.
And (3) intercepting short sequences on the 2019 novel coronavirus genome sequence in a sliding mode by using a window with the length of 15 and the step of 1 until the whole genome is traversed to generate 392 short sequence sets of the 2019 novel coronavirus genome.
And thirdly, filtering the short sequence set.
And comparing the short sequence set with a human reference genome, filtering the short sequences when the short sequences are 100% matched with the human reference genome sequence, specifically comparing the remaining specific short sequence set at an NCBI website, filtering sequences which are 100% matched with other pathogens in an NCBI database, and remaining 180 specific sequence sets.
And fourthly, selecting a specific sequence.
In the reverse transcription process, the specific primer is used to extend the template chain for one-chain cDNA synthesis, and the extension length is at least 1000bp, so that the specific primer with proper interval in SARS-CoV-2 genome position, about 800-1000bp interval, is selected as the candidate primer for experimental verification. The sequences of the candidate primer sets are shown in the following table.
TABLE 1 SARS-CoV-2 candidate primer set
Example 2
The amplification primer group experiment for detecting SARS-CoV-2 by using mNGS verifies (different dosage).
Firstly, a sample source.
The sample nucleic acid is verified to be positive by CFDA approved in-vitro diagnostic reagent (fluorescent quantitative PCR method), and the SARS-CoV-2 copy number is about 2 × 10 by standard curve quantification6copies/mL。
The specific calibration method comprises the following steps: the method comprises the steps of transcribing 509bp partial sequence of ORF1ab of the novel coronavirus in vitro, calculating copy number of a target fragment contained in a purified product according to the molecular weight of the sequence and the total amount of the transcribed purified product, establishing a standard curve of the copy number and the cycle number by using a medical instrument registration certificate reagent after dilution by multiple times, and then calibrating the copy number in a positive clinical sample of the novel coronavirus.
And II, preparing an experimental sample.
Because the detection limit of the metagenome for detecting the clinical respiratory tract sample RNA virus is expected to be 500-1000 copies/m L, experimental samples L1, L2 and L3 are prepared, and the titer of SARS-CoV-2 in the samples is 1 × 103copies/mL、1×104copies/m L and 1 × 105copies/mL。
The sample preparation method comprises the steps of simulating host and virus load characteristics of clinical samples, taking Hela cell RNA with the concentration of 1 ng/mu L as a dilution matrix, and taking a proper amount of positive samples to dilute the positive samples to the samples with the required copy number, wherein the proportion of the Hela cell RNA in the prepared samples is about 89% -95%.
And thirdly, designing an experiment.
The SARS-CoV-2 genome is about 30kb in length, and specific primer 32 bands (SEQ ID NO:1 to SEQ ID NO: 32 in example 1) having an interval of 800 and 1000bp at the SARS-CoV-2 genomic position were selected as a preferable primer set.
In the reverse transcription reaction, 20. mu. L of the reaction system, 1pmol of the specific primer set was suggested.
Since the titer of the virus in the clinical specimen of asymptomatic or mild infected patients is lower than the copy number claimed by the current nucleic acid detection method, the present invention is directed to the clinical specimen for detecting single copy virus, and therefore, the specific primer sets with 0.5, 1, 2, 4 and 5pmol added total amount are designed to be used as experimental groups under the conventional experimental conditions, the control group is the conventional metagenomic method, that is, the random primers are added in proper amount in the reverse transcription step, and in the present embodiment, the concentration of the random primers is 10 μ M (10 μmol/L).
And fourthly, an experimental method.
S1: the experimental design is as follows.
TABLE 2 concentration screening experiment design of SARS-CoV-2 specific primer set
S2: removing DNA in the RNA sample by using a DNA removal system, wherein the DNA removal system comprises an RNase inhibitor, DNase buffer solution and nuclease-free water; and RNA magnetic bead purification is performed after DNA removal.
S3: the pure RNA product after DNA removal is used as a template, according to the specific primer input amount in the table above, the primer group in the table below is used, and simultaneously, the random primer of 6bp oligonucleotide is added, under the condition suitable for reverse transcription, the RT-PCR technology is adopted to carry out targeted enrichment on the novel coronavirus genome, each primer is extended from the 3' end, and the first strand cDNA is synthesized.
S4: after the first-strand synthesis reaction is finished, adding a double-strand synthetase and a double-strand synthesis buffer solution, synthesizing double-strand cDNA under a proper condition, and after the reaction is finished, performing magnetic bead purification and Qubit quantification;
s5: taking 1ng of purified and quantified double-stranded cDNA as a template, performing transposase library construction (Norzan, TN501), namely performing fragmentation and adaptor addition in a transposase system, adding a fragmentation reaction termination solution after the fragmentation is finished, and then adding a labeled primer to perform PCR amplification of the library;
s6: purifying the amplified library to obtain a library sequenced on an Illumina sequencing platform, and sequencing the constructed library on the Illumina platform;
s7: removing host sequences by a bioinformatics analysis method, and analyzing to obtain the detected reads number and genome coverage information of the novel coronavirus.
Fifth, experimental results
Testing the concentration of the primers, testing 5 concentration gradients, and comparing the pathogen detection capability and the genome coverage;
TABLE 3 number of SARS-CoV-2 detected sequences and genome coverage
The above genome coverage means: the obtained sequence results are detected to account for the whole genome.
From the above results, the random primers and the specific primers were added to the one-strand synthesis reaction system in a total amount of 4pmol, and the maximum detection sensitivity was obtained, which was 10 to 15 times higher than that of the control group; genome coverage is more uniform.
Example 3
The mNGS detects the experimental verification of the amplification primer group of SARS-CoV-2 (different primer matching).
One, sample source and sample preparation were the same as in example 2.
Secondly, under the conditions of the primer set in example 2, half of the number of primer species is increased or decreased, and even genome coverage is considered when the number is increased or decreased, and the experimental design is as follows:
TABLE 4 SARS-CoV-2 specific primer combination confirmation design
Note: the "16 pieces of specific primers" in the above table refer to the sequences of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 in Table 1 of example 1;
the "32 specific primers" in the above table refer to the sequences of SEQ ID NOS: 1-32 in Table 1 of example 1;
the "48 pieces of specific primers" in the above table refer to the sequences of SEQ ID NOS: 1-48 in Table 1 of example 1.
Thirdly, the method and the steps similar to those of the example 2 are adopted to carry out the transcriptome library construction, the sequencing and the biological information analysis, and the detection results are shown in the following table:
TABLE 5 number of SARS-CoV-2 detected sequences and genome coverage
| Laboratory numbering | Sequence number (RPM) | Genome coverage |
| Y19 | 1615 | 27015/29903 |
| Y20 | 1035 | 27368/29903 |
| Y21 | 1092 | 27685/29903 |
| Y22 | 141 | 6946/29903 |
| Y23 | 14936 | 27012/29903 |
| Y24 | 16596 | 27435/29903 |
| Y25 | 15881 | 28018/29903 |
| Y26 | 2448 | 27742/29903 |
| Y27 | 100818 | 29714/29903 |
| Y28 | 125673 | 29831/29903 |
| Y29 | 84129 | 29864/29903 |
| Y30 | 10633 | 27692/29903 |
Fourth, result analysis
According to the results in the table, when the specific primers are added in an amount of 4pmol, the number of the specific primer pairs is within a certain range, and the detection sensitivity of SARS-CoV-2 is not affected, but the decrease of the number of the primer pairs affects the uniformity of the detected sequences of pathogens, and the increase of the number of the primer pairs does not significantly improve the detection sensitivity and the genome coverage.
Therefore, 32 SARS-CoV-2 specific primers were put into a single-strand synthesis reaction system in an amount of 4pmol, and the detection sensitivity and genome coverage were improved.
Example 4
And (5) verifying the methodology.
First, the sample source was the same as in example 2.
Second, design of experiment
Diluting SARS-CoV-2 positive clinical sample nucleic acid with fixed value to 3,000copies/m L with Hela cell nucleic acid of 1 ng/mu L, diluting the concentration gradient to 1,000, 300, 100, 30, 10 and 1copies/m L, carrying out 3 times repeated detection on each concentration sample according to the specified detection limit research method, detecting positive according to the detection result of more than or equal to 3, and preliminarily determining the minimum detection limit concentration.
And setting positive samples with the concentration of +/-3 times copies/m L according to the preliminarily determined minimum detection limit concentration, and repeatedly detecting each sample for 20 times to prepare the minimum detection limit by using the minimum concentration value with the detection rate of not less than 95%.
Thirdly, the method and the steps similar to those of the example 2 are adopted to carry out the transcriptome library construction, the sequencing and the biological information analysis, and the detection results are shown in the following table:
TABLE 6 detection limit prescreening
TABLE 7 detection Limit validation
Fourth, analysis of experimental results
The above experimental results were analyzed.
TABLE 8 preliminary determination of minimum detection Limit result data
| SARS-CoV-2 titer (copies/m L) | Positive rate of SARS-CoV-2 |
| 3,000 | 100％ |
| 1,000 | 100％ |
| 300 | 100％ |
| 100 | 100％ |
| 30 | 33％ |
| 10 | 0％ |
| 3 | 0％ |
| 1 | 0％ |
As can be seen from the above table, the lowest detection limit of SARS-CoV-2 is around 100copies/m L.
TABLE 9 data of minimum detection limit confirmation results
| SARS-CoV-2 titer (copies/m L) | Positive rate of SARS-CoV-2 |
| 300 | 100％ |
| 100 | 95％ |
| 30 | 40％ |
As can be seen from the above table, the lowest detection limit of SARS-CoV-2 was determined to be 100copies/m L.
Example 5
Comparing SARS-CoV-2 detection methodology.
Since the outbreak of the novel coronavirus, the national drug administration carries out emergency examination and approval work on reagents and medicines related to the detection, treatment and prognosis of the novel coronavirus and the like. Currently, about 20 units of detection reagents are available, and most of the detection reagents are nucleic acid molecular diagnostic reagents based on PCR. The low viral load of asymptomatic infected persons increases the sensitivity requirements for detection reagents. The detection sensitivity advantages of the methods of the invention were analyzed in comparison to approved reagents.
First, the sample source was the same as in example 2.
Second, design of experiment
A defined amount of SARS-CoV-2 positive clinical specimen nucleic acid was diluted with 1 ng/. mu. L Hela cell nucleic acid to 3,000copies/m L, 3-fold concentration gradient dilutions to 1,000, 300, 100, 30copies/m L the following 4 sets of experiments were performed:
control group 1: using a novel coronavirus detection kit 1 (fluorescent quantitation method) approved by the national drug administration, and performing 3 parallel tests on the 5 samples according to an operation instruction;
control group 2: using a novel coronavirus detection kit 2 (fluorescence quantitative method) approved by the national drug administration, performing 3 parallel tests on the 5 samples according to an operation instruction and judging whether the samples are positive or negative;
experimental groups: performing transcriptome library construction, sequencing and biological information analysis and judging negative and positive according to the similar method and steps of the example 2;
control group 3: preparing double-stranded cDNA according to the method of example 2, using the cDNA as a template, performing 3 parallel tests using a novel coronavirus detection kit 1 approved by the national drug administration, and determining whether the test is positive or negative;
control group 4: double-stranded cDNA was prepared according to the method of example 2, and 3 parallel tests were carried out using the cDNA as a template and a novel coronavirus detection kit 2 approved by the national drug administration and positive and negative were judged.
Third, analysis of experimental results
TABLE 10 data of minimum detection limit confirmation results
As is clear from the above result table, the detection sensitivity of the present invention is superior to that of the detection method based on the fluorescence quantitative method. When the specific primers of the present invention are used to synthesize double-stranded cDNA and then the double-stranded cDNA is detected by a fluorescence quantitative method, the detection sensitivity is not equal to that of the present invention, because the fluorescence quantitative PCR method detects a single fragment, and even if the same pathogen is detected by multiplex PCR, only mutual verification can be performed, but the sensitivity of the method cannot be increased.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Sequence listing
<110> Guangzhou micro-distant Gene technology Co., Ltd
Shenzhen micro-remote medical science and technology Limited
Guangzhou micro-distance medical laboratory Co., Ltd
Guangzhou micro-distance medical instruments Ltd
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tttgtcacgc actca 15
<210>36
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
ataaccactt acccg 15
<210>37
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
attaacctcc acacg 15
<210>38
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
acttatagag tacac 15
<210>39
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
attacctctt acgca 15
<210>40
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
gccgttgcca catag 15
<210>41
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
tatacgccaa cttag 15
<210>42
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
aatagccgcc actag 15
<210>43
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
accgcataca gtctt 15
<210>44
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
ctcgcatagt gtata 15
<210>45
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gtacgtgcat ggatt 15
<210>46
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
gatatgtacg accct 15
<210>47
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
tctaagcaca cgcct 15
<210>48
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
taatcggagc tgtga 15
Claims (10)
1. An amplification primer set for detecting SARS-CoV-2 by using mNGS, which is characterized by comprising: random primer and SARS-CoV-2 specific primer, the random primer is 5-7bp oligonucleotide, the SARS-CoV-2 specific primer is 10-20bp oligonucleotide designed for SARS-CoV-2 sequence.
2. The mNGS amplification primer set for detecting SARS-CoV-2 as claimed in claim 1, wherein the random primer is an oligonucleotide of 6bp and the SARS-CoV-2 specific primer is an oligonucleotide of 15bp designed for SARS-CoV-2 sequence.
3. The mNGS detection amplification primer set for SARS-CoV-2 according to claim 2, wherein the SARS-CoV-2 specific primer is selected from at least one of the following primers:
4. the mNGS detection SARS-CoV-2 amplification primer set according to claim 3, wherein the SARS-CoV-2 specific primer is SEQ ID NO:1 to SEQ ID NO: 32 in a primer set.
5. The mNGS amplification primer set for detecting SARS-CoV-2 as claimed in any of claims 1-4, wherein the working concentration of the SARS-CoV-2 specific primer is 4 ± 0.5 pmol/20 μ l.
6. Use of the amplification primer set for detecting SARS-CoV-2 of the mNGS of any one of claims 1 to 5 in the preparation of a reagent and/or a device for detecting SARS-CoV-2.
7. A kit for detecting SARS-CoV-2, comprising the amplification primer set for detecting SARS-CoV-2 of the mNGS of any one of claims 1 to 5.
8. A kit for detecting SARS-CoV-2 according to claim 7, wherein the kit is for detecting a nasal swab, a pharyngeal swab, sputum or an alveolar lavage sample.
9. A method for detecting SARS-CoV-2 for scientific research purposes, comprising the steps of:
s1: taking a biological sample, and extracting RNA to obtain an RNA sample;
s2: removing DNA in the RNA sample by using a DNA removal system to obtain an RNA pure product;
s3: using the pure RNA as a template, performing targeted enrichment on SARS-CoV-2 genome by using the amplification primer set according to any one of claims 1 to 5 under reverse transcription conditions by using RT-PCR technology, wherein each primer is extended from the 3' end to synthesize first strand cDNA;
s4: after the first-strand synthesis reaction is finished, adding a second-strand synthetase and a second-strand synthesis buffer solution to synthesize double-strand cDNA;
s5: using double-stranded cDNA as a template, building a transposase library, and performing PCR amplification on the library;
s6: purifying the amplified library, and sequencing by using a sequencing platform;
s7: removing the detected host sequence by bioinformatics analysis method to obtain SARS-CoV-2 genome information.
10. The method for detecting SARS-CoV-2 according to claim 9,
in the step S2, the DNA removal system includes an rnase inhibitor, dnase, a dnase buffer solution, and nuclease-free water; removing DNA and then purifying magnetic beads;
in the step S4, after the reaction of synthesizing the double-stranded cDNA is completed, magnetic bead purification and Qubit quantification are performed;
in step S5, 1ng of the purified and quantified double-stranded cDNA is used as a template, and the transposase library construction method specifically comprises: fragmenting and adding a linker by adopting a Norzan TN501 transposase system, adding a fragmentation reaction termination solution after the fragmentation is finished, and then adding a labeled primer for carrying out PCR amplification of the library.
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