CN113186348B - Primer combination and method for detecting common respiratory viruses - Google Patents

Abstract

The invention discloses a primer combination and a method for detecting common respiratory viruses, wherein the primer combination comprises two or more than two pairs of 12 pairs of primers for detecting 15 common respiratory viruses. The detection method provided by the invention is used for carrying out high-throughput sequencing on the sequence of the sample to be detected, which is obtained by capturing, and is combined with a bioinformatics analysis method, and comparing the sequence with the characteristic sequences of the nucleic acids in the specific areas of the 15 common respiratory viruses, so that whether the sequence of the sample to be detected is matched with the common 15 viruses can be accurately judged, and the detection method has the advantages of high accuracy, high flux and the like.

Description

Primer combination and method for detecting common respiratory viruses

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a primer combination and a method for detecting common 15 respiratory viruses.

Background

Respiratory viruses, which mainly invade the respiratory tract and proliferate on mucosal epithelial cells of the respiratory tract to cause local infection of respiratory tract or lesions of tissues and organs other than the respiratory tract, include common influenza viruses, human metapneumoviruses, syncytial viruses, parainfluenza viruses, coronaviruses, and the like. It is counted that about 90% to 95% of acute respiratory infections are caused clinically by this type of respiratory virus.

In recent years, new pathogenic respiratory viruses, such as metapneumoviruses, coronaviruses, bocaviruses and the like, are continuously discovered, and form a great threat to human health. Most of acute respiratory tract infections have the characteristics of short incubation period, urgent onset, strong infectivity, quick transmission and the like, and can often cause higher morbidity and mortality, especially the acute respiratory tract infections are influenced by environmental changes and ecological balance destruction, viruses are easy to generate various variations, sudden outbreaks are frequent, the illness is dangerous, and the mortality is higher. However, the infection symptoms caused by respiratory viruses are often similar, and it is difficult to make clear diagnosis by clinical symptoms and conventional detection methods.

At present, the commonly adopted method for detecting the respiratory viruses is fluorescence quantitative PCR (polymerase chain reaction) and the like, the methods do not obtain the sequences of the viruses, and if the viruses mutate by a few bases, the missed detection conditions such as fluorescent signal disappearance and the like can be caused. Therefore, a method with higher sensitivity, higher specificity and wider detection range is urgently needed for detection, so that various respiratory viruses can be well distinguished.

Disclosure of Invention

Based on the above, the invention provides a primer combination for detecting common respiratory viruses, wherein the primer combination can detect common 15 respiratory viruses at most once.

The specific technical scheme for realizing the aim of the invention is as follows:

A primer combination for detecting common respiratory viruses comprises primer pairs SEQ ID No. 1-2 for influenza A virus detection, primer pairs SEQ ID No. 3-4 for influenza B virus detection, primer pairs SEQ ID No. 5-6 for human rhinovirus detection, primer pairs SEQ ID No. 7-8 for human metapneumovirus detection, primer pairs SEQ ID No. 9-10 for parainfluenza virus type 1 detection, primer pairs SEQ ID No. 11-12 for parainfluenza virus type 2 detection, primer pairs SEQ ID No. 13-14 for parainfluenza virus type 3 detection, primer pairs SEQ ID No. 15-16 for OC43 coronavirus and 229E coronavirus detection, primer pairs SEQ ID No. 19-20 for HKU1 coronavirus and NL63 coronavirus detection, primer pairs SEQ ID No. 21-24 for syncytial virus type A and B coronavirus detection, and novel primer pairs ncov-ncov for both or both of these primer pairs.

In some of these embodiments, the primer combination is at a working concentration of 10nM to 10uM.

In some of these embodiments, the primer combination is operated at a concentration of 100nM to 10uM.

The invention also aims to provide a detection method of the common respiratory viruses, which has the characteristics of high sensitivity, strong specificity and high flux.

The specific technical scheme for realizing the aim of the invention is as follows:

A detection method of common respiratory viruses comprises the following steps:

(1) Mixing two or more than two pairs of 12 pairs of primers for detecting common respiratory viruses to prepare mixed primers; the working concentration of the mixed primer is 10nM-10uM;

The 12 pairs of primers are respectively as follows: primer pairs for influenza A virus detection SEQ ID No. 1-2, primer pairs for influenza B virus detection SEQ ID No. 3-4, primer pairs for human rhinovirus detection SEQ ID No. 5-6, primer pairs for human metapneumovirus detection SEQ ID No. 7-8, primer pairs for parainfluenza virus type 1 detection SEQ ID No. 9-10, primer pairs for parainfluenza virus type 2 detection SEQ ID No. 11-12, primer pairs for parainfluenza virus type 3 detection SEQ ID No. 13-14, primer pairs for parainfluenza virus type 4 detection SEQ ID No. 15-16, primer pairs for OC43 coronavirus and 229E coronavirus detection SEQ ID No. 17-18, primer pairs for HKU type 1 coronavirus and NL63 coronavirus detection SEQ ID No. 19-20, primer pairs for syncytial virus type A and B detection SEQ ID No. 21-20122, and novel primer pairs for parainfluenza virus type ncov detection SEQ ID No. 24-ncov;

(2) Extracting RNA in a sample to be detected, and performing reverse transcription to obtain cDNA;

(3) Performing a first PCR amplification reaction by using the cDNA of the step (2) as a template and the mixed primer of the step (1) as a primer;

(4) Recovering and purifying the first PCR amplification product;

(5) Connecting the first PCR amplification product purified in the step (4) with different Barcode sequences according to different samples to be detected, connecting a sequencing joint at the same time, and carrying out a second PCR amplification reaction by taking the first PCR amplification product as a template to purify the amplification product;

(6) Mixing and sequencing all the products of the second PCR amplification reaction in the step (5); and identifying the type of the respiratory viruses in the sample to be detected.

In some embodiments, the reaction system of the first PCR amplification reaction in step (3) is: in a 30ul reaction system, 10-14 ul of cDNA of a sample to be detected, 6-10 ul of mixed primer and 8-12 ul of PCR enzyme.

In some embodiments, the reaction conditions of the first PCR amplification reaction in step (3) are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 20s, extension at 60℃for 4min,25 cycles; extending at 72 ℃ for 4min; preserving heat at 10 ℃.

In some of these embodiments, the purifying in step (4) comprises the steps of:

a) Adding 0.4-0.6 times of DNA purified magnetic beads with the original PCR reaction volume into the PCR amplification product, blowing up and down for 10-15 times by a pipette, and standing for 1-2 minutes at room temperature;

b) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and taking supernatant;

c) Adding 0.5-0.8 times of DNA purified magnetic beads with the original PCR reaction volume into the supernatant, blowing up and down for 10-15 times by a pipette, and standing for 1-2 minutes at room temperature;

d) The beads were adsorbed by a magnetic rack until the solution was clear, and the supernatant was discarded.

In some embodiments, the reaction system of the second PCR amplification reaction in step (5) is: in a 30ul reaction system, 16-20 ul of purified first PCR amplification product, 8-10 ul of PCR enzyme, 2× PCR PRIMERF 0.5, 0.5-1.5 ul of PCR enzyme and 2× PCRBarcodeXX R0.5, 0.5-1.5 ul of PCR enzyme are purified, wherein the BarcodeXX means that different samples to be detected use different barcode primers.

In some of these embodiments, the reaction conditions of the second PCR amplification reaction in step (5) are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, elongation at 72℃for 1min, 6-8 cycles; extending at 72 ℃ for 5min, and preserving heat at 10 ℃.

In some of these embodiments, the purifying in step (5) comprises the steps of:

a) Adding 0.8-1.2 times of DNA purified magnetic beads into the PCR amplification product, blowing uniformly up and down by a pipettor, and standing for 1-2 minutes at room temperature;

b) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and discarding the supernatant;

c) Adding 35-45 ul BW08 into the magnetic beads, uniformly swirling, and standing at room temperature for 1-2 min;

d) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and discarding the supernatant;

e) 80-120 ul of 80% ethanol is added, magnetic beads are repeatedly adsorbed on different two sides by a magnetic rack, and the supernatant is discarded.

In some of these embodiments, the identifying in step (6) employs a bioinformatics analysis method that is: comparing the original fastq file with the amplified region sequences of the respiratory viruses with the sequence numbers shown as SEQ ID No. 25-36 by bwa software; and filtering the number of reads with the comparison accuracy rate of more than 90%, and calculating the number of reads of the corresponding viruses on the comparison.

Compared with the prior art, the invention has the following beneficial effects:

The inventor of the invention performs primer design by comparing and screening conserved regions and interspecific specific regions in 15 common respiratory viruses, and screens to obtain primer combinations capable of simultaneously detecting the common 15 respiratory viruses; by adopting the primer combination, the characteristic sequences of the nucleic acids in the specific areas of 15 common respiratory viruses can be specifically captured based on a multiplex PCR capturing method;

The detection method of the invention is to carry out high-throughput sequencing on the sequence of the sample to be detected obtained by capturing, and combine with a bioinformatics analysis method to compare with the characteristic sequences of the nucleic acids in the specific areas of 15 common respiratory viruses, so that whether the sequence of the sample to be detected is matched with the common 15 viruses can be accurately judged, and therefore, the detection method of the invention has the advantages of high accuracy, high flux and the like.

Drawings

FIG. 1 is a flow chart of a method of detecting a common respiratory virus according to the present invention;

FIG. 2 is a predictive view of interactions between partial primers.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless otherwise defined, 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 pertains

The meaning is generally understood by those skilled in the art. 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. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

To facilitate an understanding of the present technology, some terms and phrases are defined below.

Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present invention does not necessarily refer to the same embodiment, although it may. Furthermore, the phrase "in another embodiment" as used in the present invention does not necessarily refer to a different embodiment, although it may. Accordingly, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification, the meaning of "a", "an", and "the" include plural referents. The meaning of "in" is included "in" and "on".

The present invention will be described in further detail with reference to specific examples.

Example 1 method for detecting common respiratory viruses

Referring to fig. 1, a flowchart of a method for detecting a common respiratory virus according to the present embodiment includes the following steps:

1. Sample pretreatment

In this example, a total of 253 pharyngeal samples of known clinical outcome were selected for testing, of which 233 were samples of known infection type and the other 20 were negative control samples (Table 1).

Table 1 types and numbers of samples tested

Taking 300ul of sputum from each sample into a new 1.5ml centrifuge tube, adding 300ul of sputum digestion liquid WT, carrying out vortex mixing uniformly, incubating for 30min at 37 ℃, and carrying out vortex oscillation every 5-10min during digestion, so as to finally homogenize the sputum sample.

2. RNA extraction

The nucleic acids of each sample were extracted using a common commercial RNA extraction kit (tengen or qiagen).

(1) 200Ul of homogenized sample is taken into a new 1.5ml centrifuge tube, 20 ul of proteinase K solution is added, and the mixture is uniformly mixed;

(2) Adding 220 μl of buffer PB, fully reversing and uniformly mixing, standing at 70deg.C for 10min, clearing the solution strain, and centrifuging for a short time to remove water drops on the inner wall of the tube cover; adding 220 μl of absolute ethanol, and mixing completely;

(3) Adding the solution obtained in the last step and flocculent precipitate into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30sec, pouring out waste liquid, and placing an adsorption column CB into the collecting pipe;

(4) Adding 500 μl buffer PD to the adsorption column CB, centrifuging at 12,000rpm for 30sec, pouring out the waste liquid, and placing the adsorption column CB back into the collection tube;

(5) Adding 600 μl of the rinse solution PW to the adsorption column CB, centrifuging at 12,000rpm for 30sec, pouring out the waste liquid, and placing the adsorption column CB back into the collection tube;

(6) Placing the adsorption column CB back into a collecting pipe, centrifuging at 12,000rpm for 2min, and pouring out the waste liquid;

(7) Placing the adsorption column CB at room temperature for 3 minutes in an open mode to thoroughly dry residual rinsing liquid in the adsorption material;

(8) Transferring the adsorption column CB into a clean centrifuge tube, suspending and dripping 60 μl of eluent TE into the middle part of the adsorption film, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min, and collecting the solution into a separation tube;

(9) The solution obtained by centrifugation was transferred again to an adsorption column, and the solution was left at room temperature for 2 minutes and centrifuged at 12,000rpm for 2 minutes to recover the nucleic acid product.

3. RNA reverse transcription

Reverse transcriptase is used to reverse transcribe the extracted RNA into cDNA in preparation for the subsequent PCR reaction.

4. Primer sequence set mixing

The interaction between the primers is predicted by a bioinformatics method to adjust the primer sequence, so that the formation of dimers, hairpin structures and the like between the primers is avoided, and the amplification efficiency of the primers is ensured to be normal. FIG. 2 is a predictive of interactions prior to partial primer; it was shown that dimer_low indicates that the ability of the dimer formed between the two pairs of primers was weak, and that the amplification efficiency of the primers was ensured to be normal.

The 12 pairs of primers described in Table 2 were purified by HPLC synthesis, and 200ul of TE was added to each of the primers after 2OD synthesis, and the 12 pairs of primers were mixed into one tube to obtain a mixed primer Panel Mix.

TABLE 2 detection primer pair for common respiratory viruses

5. First round PCR reaction

And (3) taking 12ul of the sample nucleic acid cDNA to be detected in the step (3), 8ul of the mixed primer Panel Mix in the step (4), 10ul of the PCR enzyme, mixing into a 30ul amplification system, and carrying out a first round of PCR amplification reaction.

PCR amplification reaction procedure: pre-denaturation at 95℃for 3min; polymerase chain reaction amplification stage: a variability of 20s at 95℃and an extension of 4min at 60℃and 25 cycles were performed; extending at 72 ℃ for 4min, and preserving heat at 10 ℃ to finish amplification.

The first round of PCR reaction can effectively enrich target region fragments and reduce dimers or non-specifically amplified amplification products. After enrichment, the nucleic acid product is mainly the sequence of the target virus, so that a large amount of human-derived nucleic acid fragment pollution can be avoided. And purifying the obtained target region fragment by using an Ampure XP magnetic bead purification kit, removing dimers of the small fragments, and carrying out non-specific amplification without the target region, thereby improving the effective amplified fragment proportion. The specific purification steps are as follows:

a) Adding 0.5 times of DNA purification magnetic beads with the original PCR reaction volume into the PCR amplified product; blowing up and down for 10-15 times with 50ul range of a pipette to fully mix amplified products with magnetic beads, and standing for 2 minutes at room temperature;

b) Adsorbing the magnetic beads by a magnetic frame until the solution is clear; transferring the supernatant to a new EP tube by using a pipette, avoiding sucking magnetic beads and discarding the magnetic beads;

c) Adding 0.6 times of DNA purification magnetic beads with the original PCR reaction volume into the supernatant; blowing up and down for 10-15 times with 50ul range of a pipette to fully mix amplified products with magnetic beads, and standing for 2 minutes at room temperature;

d) Adsorbing the magnetic beads by a magnetic frame until the solution is clear; the supernatant was carefully removed with a pipette to avoid the attraction of magnetic beads.

6. Second round PCR reaction

And carrying out a second PCR on the target region recovery product, and connecting different sequence labels (only two bases are different between sequence notes) to different samples, and simultaneously connecting a specific sequencing joint to ensure that the amplified product can be connected to a sequencing chip.

The reaction system is as follows: the first round of PCR recovered 18ul of product, 10ul of PCR enzyme, 2X PCRPRIMER F and 2X PCRBarcodeXX R (different samples using different barcode primers) each 1ul.

PCR amplification reaction procedure: pre-denaturation at 95℃for 3min; polymerase chain reaction amplification stage: 15s of 95 ℃ variability, 15s of 58 ℃ annealing, and 1min of 72 ℃ extension, and carrying out 6-8 cycles; and (5) extending at 72 ℃ for 5min, and preserving heat at 10 ℃ to finish amplification.

The product obtained from the second round of PCR reaction was purified using the Ampure XP magnetic bead purification kit. The purified product is a constructed library, the concentration of the library is quantitatively detected, and the library concentration is greater than 1ng/ul and then the sequencing is carried out on the machine. The specific purification method is as follows:

a) Adding 0.9 times of volume of DNA purified magnetic beads into the PCR amplified product, and blowing up and down with a50 ul range of a pipette to fully and uniformly mix the recovered product with the magnetic beads; standing at room temperature for 2 minutes.

B) Adsorbing the magnetic beads by a magnetic frame until the solution is clear;

c) Carefully removing the supernatant by using a pipette, and avoiding sucking magnetic beads;

d) Adding 40ul of BW08 into the magnetic beads, uniformly swirling, and standing at room temperature for 2min;

e) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and removing the supernatant by a pipette;

f) 100ul of 80% ethanol is added, magnetic beads are repeatedly adsorbed back and forth on different two sides by a magnetic frame to fully suspend and wash the magnetic beads, and a pipettor is used for carefully removing the supernatant to avoid sucking the magnetic beads.

7. Sequencing data analysis

Different samples have different tag sequences, and can be subjected to mixed sequencing, so that the detection flux can be improved. The machine can simultaneously detect 48 samples or 96 samples at a time. And identifying the virus type in the sample to be detected through bioinformatic analysis.

Sequencing data was first prepared by conventional processing: converting an original sequencing fluorescent signal bcl file into a fastq file, and splitting different samples according to a barcode tag sequence; performing data quality filtering on the fastq file, and removing reads with Q30 ratio less than 90%; then removing reads smaller than 50bp in sequencing and sequencing errors, and removing nonspecifically amplified reads; then adopting bwa software to compare the filtered reads with template sequences of 15 respiratory viruses in table 3, filtering out reads with comparison quality value smaller than 15 and comparison accuracy smaller than 90%; the number of reads on each virus alignment was then counted.

TABLE 3 amplified regions of different respiratory viruses

8. Test results

Table 4 shows the quality control results of a portion of the samples, total pairreads is the total reads taken by each sample after the barcode splitting. high qualitypairreads (Q30) is the reads remaining after the Q value is less than Q30 is filtered out; ADAPTER FILTER and dimmerreads show ratios for filtering out dimers or shortreads. Nonspecific amplification refers to the ratio of reads filtered to remove non-specific amplification between primers; CLEAN PAIRREADS represents the number of reads left after low quality, dimer, nonspecific and some other reject filtration;

TABLE 4 quality control results for partial samples

After layer-by-layer filtration, the rest reads are aligned on a constructed reference sequence template (table 3), the reads with mismatch more than 10% are filtered out, and the number of reads aligned on the reference sequence template is counted; table 5 shows the number of reads aligned in each virus in a portion of the sample.

TABLE 5 number of reads aligned in each virus for part of the sample

It can be seen from Table 5 that 202001190001 samples were coronavirus 229E/NL63 virus infection, 202001200001 samples were negative samples, 202001200003 samples were respiratory syncytial virus A/B infection, 202001200004 samples were influenza virus A infection, and 202002200004 was novel coronavirus infection. The test results of these 5 samples are consistent with the actual clinical observations.

Table 6 shows the anastomosis of all 253 samples tested in this example.

TABLE 6 detection of anastomosis for 253 samples

In summary, out of 233 samples tested for known positive results in this example, 229 samples matched with the test results, with a sensitivity of 98%. The 20 negative control samples are all negative to detection, and the specificity is as follows: 100%.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention, which falls within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

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<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 35

cagatgcaac caacaacatt cattggtctt atttacatat aaagtttgct gaacctatca 60

gtctttttgt ctgtgatgcc gaattgtctg taacagtcaa ctggagtaaa attataatag 120

aatggagcaa gcatgtaaga aagtgcaagt actgttcctc agttaa 166

<210> 36

<211> 179

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 36

ttctgtggcc ctgatggcta ccctcttgag tgcattaaag accttctagc acgtgctggt 60

aaagcttcat gcactttgtc cgaacaactg gactttattg acactaagag gggtgtatac 120

tgctgccgtg aacatgagca tgaaattgct tggtacacgg aacgttctga aaagagcta 179

Claims (8)

1. The primer combination for detecting the common respiratory viruses is characterized by comprising primer pairs SEQ ID No. 1-2 for detecting influenza A viruses, primer pairs SEQ ID No. 3-4 for detecting influenza B viruses, primer pairs SEQ ID No. 5-6 for detecting human rhinoviruses, primer pairs SEQ ID No. 7-8 for detecting human metapneumoviruses, primer pairs SEQ ID No. 9-10 for detecting parainfluenza viruses1, primer pairs SEQ ID No. 11-12 for detecting parainfluenza viruses 2, primer pairs SEQ ID No. 13-14 for detecting parainfluenza viruses 4, primer pairs SEQ ID No. 15-16 for detecting OC43 coronaviruses and 229E coronaviruses, primer pairs SEQ ID No. 19-20 for detecting HKU1 coronaviruses and NL63 coronaviruses, and novel primer pairs SEQ ID No. 24-ncov for detecting parainfluenza viruses 21-24.

2. The primer combination for detecting common respiratory viruses of claim 1, wherein the primer combination has a working concentration of 10nM to 10uM.

3. The primer combination for detecting common respiratory viruses of claim 2, wherein the working concentration of the primer combination is 100nM-10uM.

4. A method for detecting a common respiratory virus for non-disease diagnostic purposes, comprising the steps of:

(1) Mixing 12 pairs of primers for detecting common respiratory viruses to prepare mixed primers; the working concentration of the mixed primer is 10nM-10uM; the 12 pairs of primers are respectively as follows: primer pairs for influenza A virus detection SEQ ID No. 1-2, primer pairs for influenza B virus detection SEQ ID No. 3-4, primer pairs for human rhinovirus detection SEQ ID No. 5-6, primer pairs for human metapneumovirus detection SEQ ID No. 7-8, primer pairs for parainfluenza virus type 1 detection SEQ ID No. 9-10, primer pairs for parainfluenza virus type 2 detection SEQ ID No. 11-12, primer pairs for parainfluenza virus type 3 detection SEQ ID No. 13-14, primer pairs for parainfluenza virus type 4 detection SEQ ID No. 15-16, primer pairs for OC43 coronavirus and 229E coronavirus detection SEQ ID No. 17-18, primer pairs for HKU type 1 coronavirus and NL63 coronavirus detection SEQ ID No. 19-20, primer pairs for syncytial virus type A and B detection SEQ ID No. 21-22, and novel primer pairs for coronavirus type ncov-23;

(2) Extracting RNA in a sample to be detected, and performing reverse transcription to obtain cDNA;

(3) Performing a first PCR amplification reaction by using the cDNA of the step (2) as a template and the mixed primer of the step (1) as a primer;

(4) Recovering and purifying the first PCR amplification product;

(5) Connecting the purified first PCR amplification product in the step (4) with different Barcode sequences according to different samples to be detected, connecting sequencing joints at the same time, and carrying out a second PCR amplification reaction by taking the sample as a template, so that different samples to be detected are connected with different sequence labels, and connecting a specific sequencing joint at the same time, so that the second PCR amplification reaction product can be connected to a sequencing chip; purifying the second PCR amplification reaction product;

(6) Mixing and sequencing all the products of the second PCR amplification reaction in the step (5); and identifying the type of the respiratory viruses in the sample to be detected.

5. The method according to claim 4, wherein the first PCR amplification reaction in the step (3) is performed by a reaction system comprising: in a 30ul reaction system, 8-14 ul of cDNA of a sample to be detected, 4-10 ul of mixed primers and 8-12 ul of PCR enzyme.

6. The method according to claim 4, wherein the reaction conditions of the first PCR amplification reaction in the step (3) are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 20s, extension at 60℃for 4min,25 cycles; extending at 72 ℃ for 4min; preserving heat at 10 ℃.

7. The method for detecting a common respiratory virus for non-disease diagnostic purposes according to claim 4, wherein the purification in step (4) comprises the steps of:

a) Adding 0.4-0.6 times of DNA purified magnetic beads with the original PCR reaction volume into the PCR amplification product, blowing up and down for 10-15 times by a pipette, and standing for 1-2 minutes at room temperature;

b) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and taking supernatant;

c) Adding DNA purification magnetic beads with the volume which is 0.5-0.8 times of the original PCR reaction volume into the supernatant, blowing up and down for 10-15 times by a pipette, and standing for 1-2 minutes at room temperature;

d) The beads were adsorbed by a magnetic rack until the solution was clear, and the supernatant was discarded.

8. The method for detecting a common respiratory virus for non-disease diagnostic purposes according to claim 4, wherein the purification in step (5) comprises the steps of:

a) Adding 0.8-1.2 times of DNA purified magnetic beads into the PCR amplification product, blowing the mixture up and down uniformly by a pipette, and standing the mixture at room temperature for 1-2 minutes;

b) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and discarding the supernatant;

c) Adding 35-45 ul BW08 into the magnetic beads, uniformly swirling, and standing for 1-2 min at room temperature;

d) Adsorbing the magnetic beads by a magnetic frame until the solution is clear, and discarding the supernatant;

e) Adding 80-120 ul of 80% ethanol, repeatedly adsorbing magnetic beads on different two sides by using a magnetic rack, and discarding supernatant.