Primer group, kit and method for detecting coxsackie virus group A type 6 virus whole genome and application of primer group, kit and method for detecting coxsackie virus group A type 6 virus whole genome

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a primer group, a kit and a method for detecting a coxsackie virus group A type 6 virus whole genome and application. A set of specific primer group aiming at CV-A6 whole genome sequence amplification is designed, aiming at different CV-A6 strains (different regions and different separation time), the super-multiplex PCR amplification can be realized, the whole genome sequence is further captured, the pathogen capture can be quickly realized at low cost in disease outbreak and epidemic periods, and the primer group has good practical value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a primer set, a kit, a method and an application for detecting the whole genome of Coxsackievirus A group 6 virus. Background Art

[0002] The incidence of hand, foot and mouth disease (HFMD) ranks first among Class C infectious diseases all year round and is a key infectious disease that needs to be controlled in my country. In recent years, many regions at home and abroad have reported changes in the pathogenic composition of HFMD. Coxsackievirus A6 (CV-A6) has gradually become the dominant pathogen causing HFMD. A recent report from the Chinese Center for Disease Control and Prevention showed that CV-A6 has become the main pathogen causing severe HFMD cases, and is still undergoing genetic evolution and has the risk of outbreaks. Therefore, improving the detection capability of CV-A6 is of great significance for the prevention and early warning of HFMD.

[0003] The amplicon-based next-generation sequencing (NGS) technology requires a low amount of starting DNA, and primers are designed for the target gene to achieve targeted amplification. Therefore, pathogen detection can be carried out as soon as the patient specimen is obtained, without relying on traditional virus isolation, which greatly saves detection time. Its detection speed is higher than that of first-generation sequencing, the detection cost is lower than that of third-generation sequencing, and it has high accuracy. Therefore, the successful construction and promotion of this method will have very important public health and socioeconomic significance. However, there are currently few specific NGS kits for CV-A6 on the market, and most of the existing kits target multiple pathogens of HFMD at the same time, making it difficult to accurately capture the whole genome sequence of the virus; in addition, Illumina is the global leader in gene sequencing and chip technology. As a popular and highly mature platform for second-generation sequencing, the Illumina platform occupies an advantageous position in the field of short-read sequencing. At present, most medical institutions have a complete platform for high-throughput sequencing, while the whole genome sequence capture carried out by sequencing companies is costly and time-consuming. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a primer set, a kit, a method and an application for detecting the whole genome of Coxsackievirus A group 6 virus. The present invention designs a primer set and a complete detection process specifically for amplifying the whole genome sequence of CV-A6, which can achieve super-multiplex PCR amplification for different CV-A6 strains (different regions, different isolation times), and then capture their whole genome sequences, which can quickly and low-costly capture pathogens during disease outbreaks and epidemics. Based on the above research results, the present invention is completed.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a primer set for detecting the whole genome of CV-A6 virus, wherein the primer set is used to detect the whole genome sequence of CV-A6 virus. Specifically, the primer set can amplify the complete genome sequence of CV-A6 virus.

[0007] The nucleotide sequences of the primers included in the primer set are shown in SEQ ID NO.1 to SEQ ID NO.92 (Table 1).

[0008] The second aspect of the present invention provides the use of the above primer set in the preparation of a product for determining the CV-A6 strain in hand, foot and mouth disease.

[0009] The third aspect of the present invention provides a kit for detecting the whole genome of CV-A6 virus, wherein the kit at least comprises the above-mentioned primer set.

[0010] A fourth aspect of the present invention provides use of the above primer set or the above kit in constructing a CV-A6 virus library.

[0011] A fifth aspect of the present invention provides a method for detecting the whole genome of CV-A6 virus based on second-generation sequencing, which is used for non-diagnostic purposes and comprises the following steps:

[0012] S1. Extraction and reverse transcription of genomic RNA from CV-A6 virus samples; S2. Establishment of an amplification system and amplification program for a primer set of nucleotide sequences such as SEQ ID NO.1 to SEQ ID NO.92, and amplification of the cDNA obtained by reverse transcription in S1; S3. Purification of PCR amplification products and construction of a library; S4. Computer detection; S5. Data processing; S6. Obtaining the complete gene sequence of CV-A6 virus.

[0013] The sixth aspect of the present invention provides an application of the above method in preparing a product for obtaining the complete gene sequence of CV-A6 viruses of different subtypes.

[0014] Beneficial technical effects of one or more of the above technical solutions:

[0015] (1) The present invention provides a primer set that can rapidly, at low cost, and with high throughput measure the entire CV-A6 genome.

[0016] (2) The present invention combines the high accuracy of first-generation sequencing with the high throughput of second-generation sequencing to capture the whole genome sequence of CV-A6 in strains and even clinical specimens, thereby improving the accuracy of pathogen detection and identification. Multiple samples can be tested simultaneously, reducing sequencing costs and improving sequencing efficiency.

[0017] (3) The above detection method can realize super-multiplex PCR amplification of different CV-A6 strains (different regions, different isolation times), and then capture their whole genome sequences. It can quickly and low-costly capture pathogens during disease outbreaks and epidemics, and has the possibility of being promoted throughout the province and even the country, thereby helping medical institutions such as CDCs to realize independent pathogen capture and traceability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 It is a schematic diagram of the design of the first round of primer sets in the whole genome of CV-A6 virus of the present invention;

[0020] Figure 2 It is the complete set of sequencing determination process of CV-A6 virus primer set and whole genome capture in the present invention;

[0021] Figure 3 The results of the offline operation and the mapping of the reference sequence in Embodiment 1 of the present invention are shown below;

[0022] Figure 4 is the mapping value of the offline result and the reference sequence in Example 1 of the present invention;

[0023] Figure 5 is the blast comparison result of the captured sequence in Example 1 of the present invention;

[0024] Figure 6 The results of the offline operation and the mapping of the reference sequence in Embodiment 2 of the present invention are shown below;

[0025] Figure 7 is the mapping value of the offline result and the reference sequence in Example 2 of the present invention;

[0026] Figure 8 is the blast comparison result of the captured sequence in Example 2 of the present invention;

[0027] Fig. 9 The phylogenetic tree results were constructed based on the sequencing results of Examples 1 and 2 of the present invention;

[0028] Fig.10This is the sequence comparison result between a strain sequence in the present invention and the sequencing result of the company. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0031] In a typical embodiment of the present invention, a primer set for detecting the whole genome of CV-A6 virus is provided. The primer set is used to detect the whole genome sequence of CV-A6 virus. Specifically, the present invention refers to the whole genome sequence of CV-A6 and designs 46 pairs of primers that can cover the whole genome and overlap each other head to tail in a shingled manner, and are named 1L, 1R, 2L, 2R...46L, 46R in sequence. The primers with odd numbers belong to primer set A, and the primers with even numbers belong to primer set B. The grouping strategy is as follows: Figure 1 As shown in the figure, the basis for adopting this pooling strategy is to amplify at intervals to avoid non-specific amplification caused by primers being too close to form dimers. Primers of the same group can be mixed together for PCR, and different primer groups can be amplified in different tubes. Finally, the PCR products of tubes A and B are mixed in the same tube for subsequent library construction. The above primer set can amplify the complete sequence within the primer range on the gene coding region, including any type of mutation sequence within the primer range.

[0032] In another specific embodiment of the present invention, the amplification product of each pair of newly added primers is about 400 bp.

[0033] In another specific embodiment of the present invention, primers are designed in the conserved region of the gene, and if there is a SNP at the primer position, a degenerate base primer is used.

[0034] In another specific embodiment of the present invention, the nucleotide sequences of the primers contained in the above primer set are shown in SEQ ID NO.1 to SEQ ID NO.92, and the primer set is also called the first round primer set, which is divided into primer set A and primer set B. The specific sequences are shown in Table 1.

[0035] Table 1 Primer sequences of the first round primer set

[0036]

[0037]

[0038]

[0039] In another specific embodiment of the present invention, a supplementary primer set designed for fragments that were not successfully captured is also included.

[0040] In another specific embodiment of the present invention, the nucleotide sequence of the supplementary primer set is shown in SEQ ID NO.93 to SEQ ID NO.112, and the supplementary primer set is also called the second-round primer set. The supplementary primer set is designed for 4 different fragments, and its design principle is the same as the first-round primer set. It is divided into primer set A1 and primer set B1, and the specific sequence is shown in Table 2.

[0041] Table 2 Primer sequences of the second round primer set

[0042]

[0043]

[0044] The primer set of the present invention can be used for amplifying the target fragments of the entire genome of CV-A6 virus in two PCR reaction systems.

[0045] In another specific embodiment of the present invention, library construction and on-machine detection are performed based on the above primer set to achieve simultaneous capture of the whole genome sequences of multiple strains. Specifically, the primer determination method of the above primer set is as follows: Figure 2 As shown, the steps are as follows:

[0046] (1) Acquisition of known CV-A6 genome sequences

[0047] All CV-A6 strain sequences were downloaded from NCBI. After sequence alignment using MAFFT online website, phylogenetic trees were constructed using iqTree to evaluate the relationship of all sequences.

[0048] (2) Screening template sequences for primer design

[0049] Only 1-2 strains of sequences located in the same evolutionary branch were retained, and finally strains with large sequence differences were screened out, and sequence alignment was used for subsequent primer set design.

[0050] (3) Primer set design

[0051] A. Upload the above-screened sequence alignment results (fasta format) to the PrimalScheme:primer panelsfor mμLtiplex PCR website for preliminary design of primer sets, and set the amplification target fragment size to 400bp.

[0052] B. Validation of the initial synthetic primer set. First, SnapGene was used for theoretical verification to ensure that all primers met the following conditions: ① Similar Tm values, ② Similar GC% values, and ③ Similar lengths of upstream and downstream primers in the same pair of primers. Those that did not meet the conditions were eliminated. Primers were synthesized by BGI and then used TaKaRa reverse transcription (6215A) and PCR kits (RR003A) and Novozymes library construction kit (ND617). Experimental operations were performed according to the relevant instructions, MiSeq was used for NGS sequencing, and CLC software was used for quality control and splicing of sequencing results.

[0053] After NGS evaluates the sequencing quality, the specific locations of the target fragments that could not be captured are screened out.

[0054] (4) Primer set optimization

[0055] A. Targetedly design primers for the unsuccessful capture fragments in SnapGene and supplement the primer set. At the same time, ensure that all primers have similar Tm values ​​and GC% values.

[0056] B. Repeat step (3) and evaluate the sequencing quality again until the primer set can successfully capture the entire CV-A6 genome sequence in one go.

[0057] In another specific embodiment of the present invention, there is provided the use of the above-mentioned first-round primer set and / or second-round primer set in the preparation of a product for determining the CV-A6 strain in hand, foot and mouth disease.

[0058] In another specific embodiment of the present invention, a kit for detecting the whole genome of CV-A6 virus is provided, wherein the kit at least comprises the above-mentioned first round primer set and / or second round primer set.

[0059] In another specific embodiment of the present invention, the kit further comprises reagents for multiplex PCR amplification reaction, wherein the reagents for amplification reaction comprise DNA polymerase, reaction buffer and enhancer, and the enhancer is betaine.

[0060] In another specific embodiment of the present invention, there is provided use of the above primer set or kit in constructing a CV-A6 virus library.

[0061] In another specific embodiment of the present invention, a method for detecting the whole genome of CV-A6 virus based on second-generation sequencing is provided, which is used for non-diagnostic purposes and comprises the following steps:

[0062] S1. Extraction and reverse transcription of genomic RNA from CV-A6 virus samples; S2. Design of amplification primers and establishment of primer amplification system and amplification program to amplify the cDNA obtained by reverse transcription in S1; S3. Purification of PCR amplification products and construction of libraries; S4. Computer detection; S5. Data processing; S6. Obtaining the complete genome sequence of CV-A6.

[0063] In another specific embodiment of the present invention, the nucleotide sequences of the primers in step S2 are as shown in SEQ ID NO.1 to SEQ ID NO.92.

[0064] In another specific embodiment of the present invention, the data processing in step S5 includes sorting the spliced ​​sequences of the detected CV-A6 virus samples and screening the CV-A6 virus samples whose full sequences have not been detected, and the CV-A6 virus samples whose full sequences have not been detected are re-detected using primers with nucleotide sequences such as SEQ ID NO.93 to SEQ ID NO.112.

[0065] The specific steps for data collation are:

[0066] ① Create Taxonomic Profiling Index: Download the complete genome sequences of all Coxsackie viruses from NCBI.

[0067] ② Taxonomic Profiling of offline data: Upload the sequencing result fastq file to CLC 21.0.2, compare it with the constructed index database, and screen the Coxsackie virus sequence with the highest similarity to each sequence to be tested.

[0068] ③ Sequence assembly (Map Reads to Reference): Use the sequences selected in the previous step as reference to assemble the whole genome sequence of the strain to be tested. This step will also generate a mapping report, and the example results are listed in the attached figure.

[0069] ④ Inspection and verification: mainly to check whether the sequence capture is complete, and further upload the whole genome to the NCBI official website for blast comparison to determine whether it is the CV-A6 sequence.

[0070] ⑤ Retest with targeted supplementary primer set: Based on the analysis in ④, screen samples with poorly captured fragments and retest using a supplementary primer set.

[0071] In another specific embodiment of the present invention, there is provided an application of the above method in preparing a product for obtaining the complete gene sequence of CV-A6 viruses of different subtypes.

[0072] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0073] Example 1

[0074] The whole genome of CV-A6 strain 1, which had been identified and successfully isolated earlier, was sequenced using next-generation sequencing.

[0075] Viral nucleic acid extraction: Use the viral DNA / RNA extraction kit (Cat. No.: T800) and fully automatic nucleic acid extractor of Tianlong Technology Company. According to the method provided in the kit, gently turn the kit upside down to mix the magnetic beads, add 200 μL of virus solution and 20 μL of proteinase K to the 1st and 7th columns of the kit, extract nucleic acid on the machine, and aspirate the nucleic acid in the 6th and 12th columns of the kit after the reaction is completed.

[0076] Viral nucleic acid amplification: using the reverse transcription kit PrimeScript produced by Takara TM IV1st strand cDNA Synthesis Mix (Cat. No.: 6215A) was used to reverse transcribe the extracted viral RNA; the specific reaction system and procedure are shown in Tables 3 and 4:

[0077] Table 3 Reverse transcription system

[0078] Reagents Usage 5×PrimeScript IV cDNA Synthesis Mix 4μL Random 6mers (50μM) 1μL Template RNA 15μL

[0079] Table 4 Reaction procedure

[0080]

[0081]

[0082] Add the same amount (here refers to volume, each primer is diluted according to the recommended ratio and then added to the same volume and mixed) of each tube of the first round of primers (SEQ ID NO.1 to SEQ ID NO.92) in accordance with the grouping criteria to tubes A and B respectively, and gently pipette to mix. TM (ExTaq TMVersion 2.0) (Cat. No.: RR003A) cDNA PCR amplification, the specific reaction system and steps are shown in Table 5 and Table 6:

[0083] Table 5 PCR reaction system

[0084] Reagents Usage Premix Taq(Ex Taq Version 2.0) 25μL The reverse transcription product from the previous step 9μL Primer set 5μL Sterilized water 11μL

[0085] Table 6 Reaction procedure

[0086]

[0087] Library construction and amplification: The amplification kit was purchased from VAHTS (Cat. No.: ND617-01-C1), the magnetic beads for purification were purchased from DNA Clean Beads (Cat. No.: N411), and the adapter kit was purchased from DNA Adapters set3-set6forIllnmina (Cat. No.: N805 / N806 / N807 / N808). DNA fragmentation: The amplified product was fragmented. The reaction system and procedure are shown in Tables 7 and 8.

[0088] Table 7 DNA fragmentation system

[0089] Reagents Usage Input DNA 35μL (17.5μL each from tube A and tube B) FEA Buffer 5μL FEA Enzyme Mix 10μL

[0090] Table 8 DNA fragmentation reaction conditions

[0091] Heating cover 105℃ On 37℃ 10 minutes 65℃ 30min 4℃ Hold

[0092] Adding adapters: Add adapters to the fragmented DNA. The specific reaction system and procedures are shown in Tables 9 and 10.

[0093] Table 9 DNA fragment plus adapter system

[0094] Reagents Usage Previous step product 50μL Rapid Ligation Buffer 3 25μL Rapid DNA Ligase 5μL DNA Adapter X 5μL <![CDATA[ddH 2 0]]> 15μL

[0095] Table 10 DNA fragment plus adapter reaction program

[0096] Heating cover 105℃ On 20℃ 15min 4℃ Hold

[0097] After adding the adapter, purification was performed, V (magnetic beads): V (adapter-added product) = 0.6:1. The specific steps are as follows:

[0098] ①After the magnetic beads are equilibrated to room temperature, vortex and oscillate to mix the VAHTSDNA Clean Beads; ②Pipette 60μL VAHTSDNA Clean Beads into 100μL Adapter Ligation product, vortex or use a pipette to gently blow 10 times to mix thoroughly; ③ Incubate at room temperature for 5 minutes; ④ Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clarified (about 5 minutes), carefully remove the supernatant; ⑤ Keep the PCR tube in the magnetic rack, add 200μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant; ⑥ Repeat step 5 for a total of two rinses; ⑦ Keep the PCR tube in the magnetic rack, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; ⑧ Take the PCR tube out of the magnetic rack and elute: add 22.5μL sterile ultrapure water to elute, vortex or use a pipette to gently blow to mix thoroughly, place at room temperature for 2 minutes, briefly centrifuge the PCR tube and place it on a magnetic rack to stand. After the solution is clarified (about 5 minutes), carefully transfer 20μL of supernatant to a new EP tube without touching the magnetic beads.

[0099] Library amplification, specific reaction system and procedures are shown in Tables 11 and 12.

[0100] Table 11 Library amplification system

[0101] Reagents Usage The product from the previous step purified by magnetic beads 20μL PCR Primer Mix 3for Illumia 5μL VAHTS HiFi Amplification Mix 25μL

[0102] Table 12 Library amplification reaction program

[0103]

[0104] The purification steps after library amplification are the same as those after adding adapters, except that V (magnetic beads): V (library amplification product) = 0.9:1 in ②, that is, 45μl VAHTSDNA Clean Beads are pipetted into 50μl LibraryAmplification product. After the amplified library is purified using magnetic beads, Qubit measures the concentration of the library corresponding to each strain, and the concentrations of all strain libraries to be tested are normalized and mixed into one tube for detection.

[0105] On-machine testing:

[0106] The detection instrument is produced by Illumina v2Reagent Kit (300Cycles PE).

[0107] Use Qubit to determine the concentration c (ng / μL) of each purified amplified library. The sample homogenization process requires 50 ng per library, so the volume required for each library is v=50 / c. Mix all samples in one tube, mix well, and use Qubit to determine the mixed concentration C. Then construct a 4nM library: the required mixture volume V=46.2 / C, and then add sterile water to make the total volume 50μL. Use 0.2N NaOH to denature the above 4nm library (the volume of the library and NaOH is 5μL), let it stand for 5min, add 990μL HT1, shake, and centrifuge instantly. According to the requirements of the machine (concentration 11pM, volume 600μL), draw 330μL from the above mixture, add HT1 to 600μL, and then test on the machine. The adapter sequence needs to be entered in advance (kit: VAHTSDNA Adaptersset3-set6 for Illumina) as shown in Table 13:

[0108] Table 13 Linker sequences

[0109]

[0110]

[0111] The MiSeq machine sets the sequencing program as follows: ① Library Prep Kit selects Custom, ② Index Kit selects Custom, ③ Index Reads selects 1, ④ Read Type selects Single Read, and ⑤ Read Lengths values ​​are all set to the default value of 151. This program is applicable to all experiments in the present invention.

[0112] In this example, the experimental data obtained by the specific operation of the machine for testing strain sample 1 are as follows: ① After the strain sample 1 library is amplified, its concentration c=10.9ng / μL is measured after dilution 5 times, ② the volume of the library (after dilution 5 times) required for normalization is calculated to be v=4.59μL, ③ the concentration of the sample after normalization is C=8.53ng / μL, ④ the volume of the mixture in ③ required to construct a 4nM library is V=5.42μL, and 44.58μL of sterile water is added. Then perform the subsequent operations described above. The detection results of the MiSeq sequencer show that %≥Q30=92.8%, indicating that the sequencing quality is high.

[0113] Example 2

[0114] The whole genome of CV-A6 strain 2, which had been previously identified and successfully isolated, was sequenced using next-generation sequencing, and the experimental methods and steps were the same as those in Example 1.

[0115] In this example, the experimental data obtained by the specific operation of the machine for testing strain sample 2 are as follows: ① After the strain sample 2 library is amplified, its concentration c=13.6ng / μL is measured after dilution 5 times, ② the volume of the library (after dilution 5 times) required for normalization is calculated to be v=3.68μL, ③ the concentration of the sample after normalization is C=9.23ng / μL, ④ the volume of the mixture in ③ required to construct a 4nM library is V=5.01μL, and then 44.99μL of sterile water is added. Then perform the subsequent operations in the above description.

[0116] The specific process of on-machine detection and off-machine data processing is the same as in Example 1, except that the number of primers included in the primer set is increased, that is, two rounds of primer sets (SEQ ID NO.1 to SEQ ID NO.112) are used for sequencing comparison. 98.30% of the sequences captured by the optimized primer pool can be successfully matched with the reference sequence (see Appendix). Figure 6 ), the capture efficiency is significantly improved.

[0117] Comparison results of sequencing quality before and after the supplementary primer set, mapping images, mapping values ​​and virus comparison results of Example 1 are as follows: Figure 3 , Figure 4 , Figure 5 As shown, the mapping pictures, mapping values ​​and virus comparison results of Example 2 are respectively as follows Figure 6 , Figure 7 , Figure 8 As shown, the phylogenetic tree constructed from the strains determined in Example 1-2 is as follows Fig. 9 shown.

[0118] To further test the accuracy of the sequencing results of the present invention, a CV-A6 strain was randomly selected and sent to Shanghai Berger for sequencing. Sequence 1, 2, and 3 represent the Berger sequencing results, the results of the nucleic acid sequencing using the present invention and the machine, and the results of the nucleic acid sequencing using the present invention and the hand-collected nucleic acid. The BioEdit sequence comparison results show that the sequence similarity of the three strains is 99% (see Appendix Fig.10 ), indicating that the present invention has high credibility, and the influence of machine extraction and hand extraction of nucleic acid on sequencing results is not significant. In order to improve efficiency in daily work, it is recommended to adopt the solution of machine extraction of nucleic acid.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer set for detecting the whole genome of CV-A6 virus, characterized in that: The nucleotide sequences of the primers contained in the primer set are shown as SEQ ID NO.1 to SEQ ID NO.

92.

2. The primer set according to claim 1, characterized in that The primer set also includes primers with nucleotide sequences as shown in SEQ ID NO.93 to SEQ ID NO.

112.

3. Use of the primer set according to any one of claims 1 to 2 in the preparation of a product for determining the CV-A6 strain in hand, foot and mouth disease.

4. A kit for detecting the whole genome of CV-A6 virus, characterized in that: The kit at least comprises the primer set according to any one of claims 1-2.

5. The kit according to claim 4, characterized in that The invention also comprises a multiplex PCR amplification reaction reagent, wherein the amplification reaction reagent comprises a DNA polymerase, a reaction buffer and an enhancer, and the enhancer is betaine.

6. Use of the primer set according to any one of claims 1-2 or the kit according to any one of claims 4-5 in constructing a CV-A6 virus library.

7. A method for detecting the whole genome of CV-A6 virus based on second-generation sequencing, the method is used for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Extraction and reverse transcription of genomic RNA from CV-A6 virus samples; S2, design amplification primers and establish a primer amplification system and amplification program to amplify the cDNA obtained by reverse transcription in S1; S3, purify the PCR amplification product and construct the library; S4, on-machine testing; S5. Data processing; S6. Obtain the complete genome sequence of CV-A6.

8. The method according to claim 7, characterized in that The nucleotide sequences of the primers in step S2 are shown in SEQ ID NO.1 to SEQ ID NO.

92.

9. The method according to claim 7, characterized in that The data processing in step S5 includes sorting the spliced ​​sequences of the detected CV-A6 virus samples and screening the CV-A6 virus samples whose full sequences have not been detected. The CV-A6 virus samples whose full sequences have not been detected are re-detected using primers with nucleotide sequences as shown in SEQ ID NO.93 to SEQ ID NO.

112.

10. Use of the method according to any one of claims 7 to 9 in preparing a product for obtaining the complete gene sequence of CV-A6 viruses of different subtypes.

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