The application discloses an amplification primer set for FMDV whole genome, an amplification method, a sequencing method and application thereof.

Abstract

This invention relates to the field of virus detection technology, specifically providing an amplification primer set, amplification method, sequencing method, and their applications for the whole FMDV genome. The amplification primer set includes 11 primer pairs, the sequences of the upstream and downstream primers of these 11 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO.22. The amplification primer set of this invention possesses good specificity and sensitivity, and is suitable for the whole genome amplification and sequencing of FMDV serotypes O, A, C, Asia I, SAT1, SAT2, and SAT3. The amplification method of this invention has promising applications in the identification and sequencing of foot-and-mouth disease virus. The sequencing method of this invention is compatible with next-generation sequencing platforms and nanopore sequencing platforms, has a high degree of automation, and can achieve 99% coverage for samples with a virus content of 1 copy / μL or higher, with a sequencing depth of over 1000X.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to an amplification primer set, amplification method, sequencing method, and their applications for the entire FMDV genome. Background Technology

[0002] Foot-and-mouth disease (FMD) Foot-and-Mouth Disease FMD is caused by foot-and-mouth disease virus (FMD). Foot-and-Mouth Disease Virus Foot-and-mouth disease virus (FMDV) is an acute, febrile, and highly contagious disease that primarily affects more than 30 species of cloven-hoofed animals, including cattle, pigs, sheep, and deer. Clinical features include blisters and ulcers on the oral mucosa, hooves, and udder skin. Young animals may die from myocarditis. FMDV belongs to the genus Aphthovirus in the family Picornaviridae. The virus particles are icosahedral in shape, approximately 28-30 nm in diameter, and lack an envelope. It is relatively resistant to environmental factors, rapidly inactivated below pH 6.0 and above pH 9.0, but can survive for extended periods in neutral environments and at low temperatures.

[0003] The FMDV genome is a single-stranded positive-sense RNA, approximately 8.4 kb in length. From the 5' end to the 3' end, the genome structure includes a 5' untranslated region (5'UTR), an open reading frame (ORF), and a 3' untranslated region (3'UTR). The 5' UTR contains the internal ribosome entry site (IRES), the ORF encodes a polyprotein precursor of approximately 2500 amino acids, and the 3' UTR contains a poly(A) tail. The polyprotein precursor is cleaved by viral proteases (Lpro, 2A, 3C) into structural protein regions (L, 1ABCD) and non-structural protein regions (2ABC, 3ABCD). The structural protein precursor encoded by the 1ABCD region (i.e., the P1 region) is processed by the proteases to produce VP0, VP3, and VP1. Subsequently, VP0 is further cleaved into VP2 and VP4, ultimately forming four mature structural proteins: VP4, VP2, VP3, and VP1. VP1 is the major antigenic determinant and a key target gene for serotyping and molecular epidemiological analysis.

[0004] FMDV exhibits high antigenic variability and genetic diversity, and is currently internationally recognized as belonging to seven serotypes: O, A, C, Asia I, SAT1, SAT2, and SAT3. There is no cross-immunity between serotypes, and each serotype can be further divided into multiple topotypes and lineages, resulting in a rich variety of reported strain sequence variations. Serotype O is the most widespread and prevalent globally, encompassing multiple topotypes (such as ME-SA, SEA, Cathay, Euro-SA, West Africa, East Africa, Indonesia-1, and Indonesia-2). Serotype A has the highest genetic diversity and is widely prevalent in Asia, Africa, and the Middle East. Asia I is mainly distributed in Asia. Serotype C has not been detected in natural circulation globally since 2004. SAT1, SAT2, and SAT3 are primarily confined to sub-Saharan Africa, but with increasing international trade in recent years, there is a risk of cross-regional transmission. Foot-and-mouth disease outbreaks of type O, type A, and Asia I have occurred in my country's history. In recent years, type O (Mya-98 lineage and Ind-2001 lineage) and type A (Sea-97 lineage) have been the main types.

[0005] It is worth noting that FMDV's RNA-dependent RNA polymerase (RdRp, i.e., the 3D protein) lacks proofreading function, resulting in an extremely high mutation rate (approximately 10) during viral replication. -3 ~10 -5 The substitutions / sites / replications of FMDV, coupled with the gene recombination that can occur between different serotypes or topological strains in the same host, lead to the continuous emergence of new variant strains, posing a persistent challenge to vaccine immunization and disease control. Rapid differential diagnosis, accurate serotyping, and obtaining the whole genome sequence information of FMDV-infected samples are fundamental for tracing the source of FMDV, monitoring mutations, assessing vaccine compatibility, and developing precise control strategies, playing an irreplaceable supporting role in the eradication and elimination of FMDV. Currently, laboratory detection methods for FMDV mainly include: virus isolation and culture (VI), enzyme-linked immunosorbent assay (ELISA, including solid-phase competitive ELISA and liquid-phase blocking ELISA), quantitative RT-PCR (qRT-PCR), Sanger sequencing, and metagenomic high-throughput sequencing (mNGS); among these, Sanger sequencing and metagenomic high-throughput sequencing (mNGS) are commonly used methods for obtaining the FMDV whole genome.

[0006] However, existing FMDV whole-genome targeted sequencing protocols generally suffer from the following common problems: (1) amplification of the highly structured 5' end region (including S-fragment and IRES secondary structures) is difficult, resulting in incomplete whole-genome coverage; (2) accurate amplification of the 3' end poly(A) tail region requires additional experimental steps; (3) the detection sensitivity is insufficient to handle clinical samples with low viral load; and (4) most protocols are only compatible with a single sequencing platform. Therefore, there is an urgent need to develop an FMDV whole-genome targeted enrichment sequencing protocol that can achieve complete coverage from the 5' end to the 3' end in a single experimental procedure, has ultra-high sensitivity, and is compatible with multiple sequencing platforms. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a primer set, amplification method, sequencing method, and their applications for the whole genome amplification of FMDV. The amplification primer set of the present invention possesses good specificity and sensitivity, and is suitable for the whole genome amplification and sequencing of FMDV serotypes O, A, C, Asia I, SAT1, SAT2, and SAT3. The amplification method of the present invention shows promising application prospects in FMDV sequencing. The sequencing method of the present invention is compatible with next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation and short sequencing time. For samples with a viral load of 1 copy / μL or higher, it can achieve a coverage rate of over 99%, and a sequencing depth of over 1000X.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a primer set for amplifying the whole genome of foot-and-mouth disease virus, the primer set comprising 11 pairs of primers, the sequences of the upstream and downstream primers of the 11 pairs of primers being shown in SEQ ID NO.1 to SEQ ID NO.22.

[0009] In some embodiments of the present invention, the 11 pairs of primers in the amplification primer set are divided into two groups, wherein primer set 1 consists of primers with sequence numbers shown in SEQ ID NO. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, and primer set 2 consists of primers with sequence numbers shown in SEQ ID NO. 3, 4, 7, 8, 11, 12, 15, 16, 19, 20.

[0010] Secondly, the present invention provides an amplification reagent for the whole genome of foot-and-mouth disease virus, the amplification reagent comprising the amplification primer set described in the first aspect.

[0011] In some embodiments of the present invention, the amplification reagent comprises two sets of reagents, wherein one set of reagents comprises primer set 1 as described in the first aspect, and the other set of reagents comprises primer set 2 as described in the first aspect.

[0012] Thirdly, the present invention provides an amplification kit for the whole genome of foot-and-mouth disease virus, the amplification kit comprising the amplification primer set described in the first aspect or the amplification reagent described in the second aspect.

[0013] Fourthly, the present invention provides a method for amplifying the whole genome of foot-and-mouth disease virus, the amplification method comprising amplification using the amplification primer set described in the first aspect, the amplification reagent described in the second aspect, or the amplification kit described in the third aspect.

[0014] In some embodiments of the present invention, the amplification procedure is as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, denaturation at 60°C for 30 seconds, denaturation at 72°C for 90 seconds, for a total of 25 cycles; extension at 72°C for 5 minutes.

[0015] In some embodiments of the present invention, the foot-and-mouth disease virus includes one or more serotypes of foot-and-mouth disease virus selected from serotypes O, A, C, Asia I, SAT1, SAT2 and SAT3.

[0016] Fifthly, the present invention provides the application of the amplification method described in the fourth aspect in the identification of foot-and-mouth disease virus for non-disease diagnosis and treatment purposes.

[0017] In some embodiments of the present invention, the foot-and-mouth disease virus includes one or more serotypes of foot-and-mouth disease virus selected from serotypes O, A, C, Asia I, SAT1, SAT2 and SAT3.

[0018] In a sixth aspect, the present invention provides a sequencing method for the whole genome of foot-and-mouth disease virus, the sequencing method comprising multiplex targeted amplification using the amplification primer set described in the first aspect, the amplification reagent described in the second aspect, or the amplification kit described in the third aspect.

[0019] In some embodiments of the present invention, the sequencing method includes the following steps: (1) Collect samples and extract nucleic acids from the samples; (2) The nucleic acid extracted in step (1) is reverse transcribed to obtain cDNA; (3) Use the amplification primer set described in the first aspect, or the amplification reagent described in the second aspect, or the amplification kit described in the third aspect to perform multiple targeted amplification on the cDNA obtained by reverse transcription in step (2); (4) Purify the nucleic acid of the amplification product obtained in step (3); (5) Constructing sequencing libraries and performing sequencing; (6) Bioinformatics analysis.

[0020] In some embodiments of the present invention, the foot-and-mouth disease virus includes one or more serotypes of foot-and-mouth disease virus selected from serotypes O, A, C, Asia I, SAT1, SAT2 and SAT3.

[0021] In some embodiments of the present invention, the sample in step (1) includes one or more of the following: nasal swab, throat swab, blister fluid, tissue homogenate, etc.

[0022] In some embodiments of the present invention, the method of step (1) nucleic acid extraction includes extraction using a nucleic acid extraction kit.

[0023] In some embodiments of the present invention, the method of step (2) reverse transcription includes cDNA synthesis using a reverse transcription kit.

[0024] In some embodiments of the present invention, when performing multiplex targeted amplification in step (3), the 11 pairs of primers in the amplification primer set described in the first aspect are divided into two groups and subjected to multiplex targeted amplification with the cDNA obtained by reverse transcription in step (2), wherein primer set 1 consists of primers with the sequences shown in SEQ ID NO. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, and primer set 2 consists of primers with the sequences shown in SEQ ID NO. 3, 4, 7, 8, 11, 12, 15, 16, 19, 20.

[0025] In some embodiments of the present invention, the length of the amplification product obtained by multiple targeted amplification in step (3) is 1000-2000 bp, preferably 1000-1500 bp, and more preferably about 1200 bp.

[0026] In some embodiments of the present invention, the procedure for multiple targeted amplification in step (3) is as follows: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, denaturation at 60°C for 30s, denaturation at 72°C for 90s, for a total of 25 cycles; extension at 72°C for 5min.

[0027] In some embodiments of the present invention, the reaction reagents for multiplex targeted amplification in step (3) include Pathogen DNA Multiplex PCR Mix, ddH2O, the amplification primer set described in the first aspect, and the cDNA obtained in step (2).

[0028] In some embodiments of the present invention, each 25 μL of the reaction reagent for the multiplex targeted amplification in step (3) includes 12.5 μL of Pathogen DNA Multiplex PCR Mix, 5 μL of ddH2O, 15 μL of the amplification primer set described in the first aspect, and 2.5 μL of cDNA obtained in step (2); the other 25 μL of the reaction reagent includes 12.5 μL of Pathogen DNA Multiplex PCR Mix, 5 μL of ddH2O, 25 μL of the primer set described in the first aspect, and 2.5 μL of cDNA obtained in step (2).

[0029] In some embodiments of the present invention, the Pathogen DNA Multiplex PCR Mix is ​​preferably Vetaiseq Pathogen DNA Multiplex PCR Mix produced by Beijing Genesys Pharma Co., Ltd.

[0030] In some embodiments of the present invention, the nucleic acid purification method in step (4) includes purification using purification magnetic beads.

[0031] In some embodiments of the present invention, step (5) of constructing the sequencing library is performed on a next-generation sequencing platform or a nanopore sequencing platform.

[0032] In some embodiments of the present invention, step (5) of constructing the sequencing library is performed on a second-generation sequencing platform; preferably, the construction of the sequencing library specifically includes the following steps: fragment fragmentation, end repair and dA addition, adapter ligation, library amplification, denaturation, single-strand circularization, enzyme digestion, and DNB preparation; more preferably, the step of constructing the sequencing library further includes purifying the product obtained from the adapter ligation step and / or purifying the product obtained from the library amplification step and / or purifying the product obtained from the enzyme digestion step; even more preferably, the purification includes purification using purification magnetic beads.

[0033] In some embodiments of the present invention, step (5) of constructing a sequencing library is performed on a nanopore sequencing platform; preferably, the construction of the sequencing library specifically includes the following steps: end repair and dA addition, barcoding and adapter ligation; more preferably, the step of constructing the sequencing library further includes purifying the product obtained from the barcoding and adapter ligation steps; even more preferably, the purification includes purification using purification magnetic beads.

[0034] In some embodiments of the present invention, purification using purifying magnetic beads includes: 1) Mix the product to be purified with magnetic beads, and discard the supernatant after the magnetic beads have completely adsorbed the product; 2) Rinse the magnetic beads with freshly prepared 80% ethanol aqueous solution and remove the supernatant; 3) Open the lid and dry the magnetic beads for 5-10 minutes at room temperature, add ddH2O to resuspend the dried magnetic beads, and collect the supernatant after the magnetic beads are completely adsorbed to obtain the purified product.

[0035] In some embodiments of the present invention, the volume ratio of the product to be purified to the magnetic beads in step 1) is (0.5 to 3): 1.

[0036] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0037] In some embodiments of the present invention, when using purification magnetic beads for purification, the container holding the product to be purified and the magnetic beads is placed on a magnetic rack during the purification process so that the magnetic beads can be adsorbed by magnetic force during the purification process.

[0038] In some embodiments of the present invention, the construction of sequencing libraries in step (5) is performed using a sequencing library construction kit or library preparation kit produced by Beijing Genesys Logic Co., Ltd., such as the Vetaiseq metagenomic DNA and RNA co-construction library kit (MGI), the Vetaiseq double barcode circularization kit, or the Vetaiseq CycloneSEQ G100-ER 24 barcode rapid library preparation kit.

[0039] In some embodiments of the present invention, the sequencing in step (5) is performed using sequencing kits or sequencing kits produced by Beijing Genesys Logic Co., Ltd., such as the Vetaiseq DNBSEQ-G99RS high-throughput sequencing kit (PE150) or the Vetaiseq Cyclone SEQ G100-ER sequencing kit.

[0040] In some embodiments of the present invention, the sequencing in step (5) is performed using a gene sequencer from Shenzhen BGI Genomics Co., Ltd., such as the BGI Genomics Cyclone SEQ-WT02 gene sequencer or the BGI Genomics DNBSEQ-G99RS gene sequencer.

[0041] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes data quality control, sequence alignment, result statistics, coverage assessment, variant detection, and evolutionary analysis.

[0042] In some embodiments of the present invention, the data quality control includes removing low-quality reads, adapter sequences and short fragments from the raw sequencing data; preferably, fastp / fastqc is used for data quality control of second-generation sequencing data, and nanopore sequencing data is used for data quality control. In some embodiments of the present invention, the sequence alignment is performed using BWA-MEM when analyzing second-generation sequencing data.

[0043] In some embodiments of the present invention, the sequence alignment is performed using minimap2 when analyzing nanopore sequencing data.

[0044] In some embodiments of the present invention, the statistical results include using samtools to calculate indicators such as comparison rate and coverage uniformity.

[0045] In some embodiments of the present invention, the coverage assessment includes using bamdst to calculate whole-genome coverage and sequencing depth.

[0046] In some embodiments of the present invention, the mutation detection includes SNP and InDel mutation detection using bcftools or GATK.

[0047] In some embodiments of the present invention, the evolutionary analysis includes multiple sequence alignment using MAFFT and constructing a maximum likelihood phylogenetic tree using IQ-TREE.

[0048] In a seventh aspect, the present invention provides the application of the sequencing method described in the sixth aspect in foot-and-mouth disease virus serotyping.

[0049] The beneficial effects of this invention are as follows: This invention proposes a primer set for amplifying the entire genome of foot-and-mouth disease virus (FMDV), which can cover the entire FMDV genome. The primer set of this invention has good specificity and sensitivity and is suitable for the amplification and sequencing of the entire genome of FMDV serotypes O, A, C, Asia I, SAT1, SAT2 and SAT3.

[0050] This invention also proposes amplification reagents and amplification kits including the amplification primer set of this invention, as well as a method for amplifying foot-and-mouth disease virus using the amplification primer set, amplification reagents or amplification kit of this invention; the amplification method of this invention has good application prospects in the identification and whole genome sequencing of foot-and-mouth disease virus.

[0051] This invention also proposes a sequencing method for the entire genome of foot-and-mouth disease virus. This sequencing method is compatible with both next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation and short sequencing time. On nanopore sequencing platforms, library preparation can be completed in as little as 0.5 hours, and analysis results can be obtained in 3 hours. Moreover, this sequencing method can achieve coverage of over 99% for samples with a virus concentration of 1 copy / μL or higher, with a sequencing depth exceeding 1000X. Attached Figure Description

[0052] Figure 1 The design diagram of the 11 pairs of primers of the present invention is shown.

[0053] Figure 2 The genome-wide coverage of FMDV in nucleic acid sample ZKJY-E4 obtained by the sequencing method of Example 1 is shown.

[0054] Figure 3 The genome-wide coverage of FMDV in nucleic acid sample ZKJY-E4 obtained by the sequencing method of Example 2 is shown.

[0055] Figure 4 The results of the typing of the A serotype vaccine strain were presented, which was obtained by phylogenetic tree analysis based on the whole genome after whole genome sequencing via a nanopore sequencing platform.

[0056] Figure 5 The results of the Asia I serotype vaccine strain were presented, obtained from whole-genome sequencing using a nanopore sequencing platform and phylogenetic tree analysis based on the whole genome.

[0057] Figure 6 The results of genotyping of the O serotype vaccine strain were presented, obtained from whole-genome sequencing using a nanopore sequencing platform and phylogenetic tree analysis based on the whole genome.

[0058] Figure 7 The results of the SAT1 serotype vaccine strain were presented, obtained from whole-genome sequencing using a nanopore sequencing platform and phylogenetic tree analysis based on the whole genome.

[0059] Figure 8 A primer matching heatmap of 11 primer pairs for 7 serotypes of FMDV is shown.

[0060] Figure 9 The genome-wide coverage of serotype C FMDV obtained by the simulated sequencing method in Example 4 is shown.

[0061] Figure 10 The genome-wide coverage of SAT2 serotype FMDV obtained by the simulated sequencing method in Example 4 is shown.

[0062] Figure 11 The genome-wide coverage of SAT3 serotype FMDV obtained by the simulated sequencing method in Example 4 is shown. Detailed Implementation

[0063] The following examples further illustrate the technology of the present invention. These examples are illustrative and exemplary of the present invention and do not limit the scope of the invention in any way.

[0064] Some of the experimental reagents used in the embodiments of this invention are shown in Table 1: Table 1 Experimental Reagents

[0065] Table 2 shows some of the experimental instruments and equipment used in the embodiments of this invention: Table 2 Experimental Instruments and Equipment

[0066] The experimental samples used in the embodiments of this invention are shown in Table 3: Table 3 Experimental Samples

[0067] In Table 3, DF-1 is a cell culture and served as a negative control in the experiment.

[0068] The amplification primer sets used in the embodiments of this invention are shown in Table 4: Table 4 Primer set for FMDV whole genome amplification

[0069] The design principles of the amplification primer sets in Table 4 are as follows: (1) Construction of reference sequence set: Complete genome sequences of each serotype of FMDV were downloaded from GenBank and WRLFMD databases, covering major topologies and prevalent lineages, and a multi-serotype reference sequence set was constructed. Analysis of the constructed reference sequence set showed that: the whole genome length of FMDV is about 8.4 kb, containing poly(A) tails of different lengths; the whole genome sequence identity among different serotypes is about 60% to 70%, but the 5'UTR, 3'UTR and some non-structural protein coding regions (such as the 3D region) are relatively conserved, while the structural protein coding regions (especially the GH loop of VP1) show the most dramatic variation.

[0070] (2) Multiple sequence alignment and reference sequence selection: MAFFT was used to perform multiple sequence alignment on all reference sequences, and primer binding sites were selected in the conserved regions of each serotype, while also taking into account the coverage of variant regions.

[0071] (3) Swaminated primer design: Automated primer design was performed using Primal Scheme (https: / / primalscheme.com), setting the amplicon size to approximately 1200 bp, resulting in 7 primer pairs covering the entire FMDV genome. Based on the automated design results, the coverage of the 5' and 3' ends of the genome was manually checked, and additional end primers were added, resulting in 2 primer pairs covering the entire FMDV genome. Two additional primer pairs covering the entire FMDV genome were added for STA1, SAT2, and SAT3. Finally, 11 primer pairs covering the entire FMDV genome were obtained. The design diagram of the 11 primer pairs is shown below. Figure 1 As shown.

[0072] Specifically, 5' and 3' end specific primer designs were employed: Targeting the highly structured S-fragment (containing tandem repeat sequences forming complex secondary structures) and IRES region at the 5' end of the FMDV genome, specific targeting primers were designed upstream of the 5' end. By optimizing primer length and annealing temperature, it was ensured that the primers could effectively bind to and amplify this highly structured region. Considering the monotypic nature of the 3' end poly(A) tail region, poly-T anchoring primers (i.e., 3' end primer sequences consisting of continuous T bases or poly-T variants containing degenerate bases) were designed to specifically anneal to the poly(A) tail. Simultaneously, a specific forward primer located in the conserved region of the 3' UTR was designed upstream to pair with it, thereby achieving specific amplification of the 3' end poly(A) region in a multiplex PCR system. The 5' end specific primers and 3' end poly-T anchoring primers were integrated into primer pool 1 and primer pool 2, respectively, and together with the remaining 9 pairs of wavy primers, formed a complete primer set of 11 pairs, achieving complete coverage of the entire FMDV genome from the 5' end to the 3' end in a single multiplex PCR process.

[0073] (4) Degenerate base optimization: For primer binding sites with large sequence differences among the seven serotypes, IUPAC degenerate bases (such as R, Y, M, K, W, S, etc.) are introduced to improve the universality of primers for different serotypes and topological strains. At the same time, the degeneracy of each primer is controlled to not exceed 8 times to avoid excessive primer set complexity.

[0074] (5) Primer specificity verification: The designed primer sequences were aligned to the NCBI nt database using BLAST to confirm that the primers specifically target the FMDV genome and do not cross-react with the host genome (cattle, pigs, sheep and other cloven-hoofed animals).

[0075] Example 1 (Next-Generation Sequencing) The experimental samples in this embodiment were: four nucleic acid samples ZKJY-E1, ZKJY-E2, ZKJY-E3, and ZKJY-E4 extracted from FMDV quality control materials and FMDV vaccine; ZKJY-E8, ZKJY-E9, ZKJY-E10, ZKJY-E11, and ZKJY-E12 were all obtained by diluting ZKJY-E4, with nucleic acid concentrations of 10000 copies / μL, 1000 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; DF-1 cell culture was used as a negative control to obtain sample DF-1; a total of 10 samples were used, with 3 replicates for each sample, for a total of 30 samples.

[0076] The samples ZKJY-E1, ZKJY-E2, and ZKJY-E3 were obtained as follows: 100 μL of FMDV quality control material was used to extract nucleic acids using the Vetaiseq DNA and RNA Co-extraction Kit (magnetic bead method) and the TGuide S16 fully automated nucleic acid extraction and purification instrument. The nucleic acids were then reconstituted using 90 μL of Elution Buffer to obtain a nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using the QuantStudio 1Plus real-time fluorescence quantitative PCR instrument, and the Ct value of the sample was obtained.

[0077] The ZKJY-E4 sample was obtained as follows: 100 μL of FMDV vaccine was used to extract nucleic acid using the Vetaiseq DNA and RNA Co-extraction Kit (magnetic bead method) and the TGuide S16 fully automated nucleic acid extraction and purification instrument. The nucleic acid was then reconstituted using 90 μL of ELution Buffer to obtain a nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using the QuantStudio 1Plus real-time fluorescence quantitative PCR instrument, and the Ct value of the sample was obtained.

[0078] The copy number of ZKJY-E4 samples was calculated using the Ct value of the ZKJY-E4 samples. Then, the ZKJY-E4 samples were diluted to 10000 copies / uL, 1000 copies / uL, 10 copies / uL, and 1 copy / uL to obtain nucleic acid samples ZKJY-E8, ZKJY-E9, ZKJY-E10, ZKJY-E11, and ZKJY-E12.

[0079] The nucleic acid purity of the above nucleic acid samples was determined using a NanoDrop micro spectrophotometer, and the nucleic acid concentration of the above nucleic acid samples was determined using a Qubit fluorometer.

[0080] The sequencing experiment process is as follows: 1. Reverse transcription: cDNA synthesis was performed on the sample RNA using the Vetaiseq reverse transcription kit, as detailed below: Take the experimental sample (RNA), prepare the reagents according to Table 5, vortex and mix (use a VORTEX-5 vortex mixer to vortex and mix, the same below), and briefly centrifuge. Place it in a PCR instrument (MiniAmp Plus Thermal Cycle PCR, the same below) and perform the PCR reaction according to the program in Table 6.

[0081] Table 5. Reagents and dosages for reverse transcription

[0082] Table 6. PCR reaction procedure for reverse transcription

[0083] 2. Targeted amplification of the entire FMDV genome Using the cDNA synthesized in step 1 as a template, amplification reagents were prepared according to Table 7 using the 11 primer pairs (SEQ ID NO.1~SEQ ID NO.22) listed in Table 4. The mixture was vortexed, briefly centrifuged, and then placed in a PCR instrument for FMDV whole-genome multiplex targeted amplification according to the procedure in Table 8. The reagents in Table 7 consist of two different sets of amplification reagents: one set containing 12.5 μL of Vetaiseq Pathogen DNA Multiplex PCR Mix, 5 μL of ddH2O, primer set 1, and 2.5 μL of the cDNA synthesized in step 1; the other set containing 12.5 μL of Vetaiseq Pathogen DNA Multiplex PCR Mix, 5 μL of ddH2O, primer set 2, and 2.5 μL of the cDNA synthesized in step 1. Both sets of amplification reagents were used for FMDV whole-genome multiplex targeted amplification according to the procedure in Table 8.

[0084] Table 7. Reagents and dosages for FMDV whole genome amplification.

[0085] Table 8. Reaction Procedure for FMDV Whole Genome Amplification

[0086] After the multiplex targeted amplification reaction was completed, 25 μL each of the amplification products containing primer set 1 and primer set 2 were combined into one tube. 40 μL of Vetaiseq DNA CleanBeads were added to the tube, and the mixture was incubated at room temperature for 5 min. The tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed, after which the supernatant was discarded. Next, 200 μL of freshly prepared 80% ethanol aqueous solution was added, and the mixture was allowed to stand for 30 s, after which the supernatant was discarded. Then, another 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube, and the mixture was allowed to stand for 30 s, after which the supernatant was discarded. Finally, the magnetic beads were left to dry at room temperature for 8 min, and 40 μL of... The dried magnetic beads were resuspended in ddH2O, removed from the magnetic rack and incubated at room temperature for 5 min, then transferred back to the magnetic rack and allowed to stand for 5 min. After the magnetic beads were completely adsorbed, the supernatant was aspirated and transferred to a 0.2 mL centrifuge tube. The purity of the nucleic acid was determined using a NanoDrop micro spectrophotometer, and the nucleic acid concentration was determined using a Qubit fluorometer. The supernatant was then temporarily stored at -20℃ for later use.

[0087] 3. Library Construction The library was constructed using the Vetaiseq Metagenomic DNA and RNA Co-construction Library Kit (MGI). The specific steps are as follows: 3.1. Fragment breaking, end-of-segment repair, and dA addition Mix the amplification product obtained in step 2 with other components according to the amounts in Table 9 (the amount of double-stranded DNA is 50 ng, and its volume is calculated based on the concentration obtained in step 2), vortex to mix, and briefly centrifuge. Place the mixture in a PCR instrument and perform the PCR reaction according to the program in Table 10.

[0088] Table 9. Reagents and dosages for fragment fragmentation, end-effector repair, and dA addition.

[0089] Table 10 PCR reaction procedures for fragment fragmentation, end repair, and dA addition.

[0090] Note: The reaction time at 30℃ is determined by the break length. For example, when the sequencing strategy is PE150, the break time is 3 min.

[0091] 3.2. Connector Connection Prepare the reagents according to Table 11 and add 40 μL of the prepared reagents to the reaction tube after the reaction in step 3.1. Shake to mix and centrifuge briefly. Place the tube in a PCR instrument and perform the PCR reaction according to the procedure in Table 12.

[0092] Table 11. Reagents and dosages for connector connection.

[0093] Table 12 PCR reaction procedures for adapter ligation

[0094] After the PCR reaction program was completed, 60 μL of Vetaiseq DNA Clean Beads was added to the reaction tube and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then discarded. Next, 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. Then, another 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. The magnetic beads were then dried at room temperature for 8 min. 20 μL of ddH2O was added to resuspend the dried magnetic beads. The magnetic rack was removed and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then aspirated and transferred to a new tube, yielding 20 μL of ligation product.

[0095] 3.3. Library Augmentation Prepare the reagents according to Table 13, where the MGI library amplification primers (PE)* are derived from the Vetaiseq metagenomic DNA and RNA co-construction library kit (MGI); then vortex to mix and briefly centrifuge, and place in a PCR instrument to perform the PCR reaction according to the procedure in Table 14.

[0096] Table 13 Reagents and dosages for library amplification

[0097] Table 14 PCR reaction procedure for library amplification

[0098] After the PCR reaction program was completed, 45 μL of Vetaiseq DNA Clean Beads was added to the reaction tube and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then discarded. Next, 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. Then, another 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. The magnetic beads were then dried at room temperature for 8 min. 20 μL of ddH2O was added to resuspend the dried magnetic beads. The magnetic rack was removed and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then aspirated and transferred to a new tube, resulting in a 20 μL library.

[0099] 3.4. Transgender The products obtained from ten samples (ZKJY-E1, ZKJY-E2, ZKJY-E3, ZKJY-E4, ZKJY-E8, ZKJY-E9, ZKJY-E10, ZKJY-E11, ZKJY-E12, and DF-1) after fragmentation and end repair, dA addition, adapter ligation and product purification, library amplification and product purification were mixed at equal mass using a DNA mass meter. Each sample was then mixed into a 1.5 mL low-adsorption tube, and water was added to bring the total volume to 34 μL. Reagents were then prepared according to Table 15 (where the oligonucleotides were derived from the Vetaiseq double barcode cyclization kit). The mixture was vortexed, briefly centrifuged, and incubated in a PCR instrument at 98°C for 3 min. Immediately afterward, the mixture was placed on ice for 2 min and briefly centrifuged again.

[0100] Table 15 Denaturing reagents and dosages

[0101] 3.5. Single-chain cyclication Prepare the reagents according to Table 16 (wherein, the ligase and ligase buffer are from the Vetaiseq double barcode cyclization kit), and add 20 μL of the prepared reagents to the reaction tube after the reaction in step 3.4. Shake to mix and centrifuge briefly, then place in a PCR instrument to perform the PCR reaction according to the program in Table 17.

[0102] Table 16 Reagents and dosages for single-chain cyclization

[0103] Table 17 PCR reaction procedure for single-stranded circularization

[0104] 3.6. Enzymatic digestion Prepare the reagents according to Table 18 (where the digestive enzyme and digestive enzyme buffer are from the Vetaiseq double barcode cyclization kit), and add 10 μL of the prepared reagents to the reaction tube after the reaction in step 3.5. Shake to mix and centrifuge briefly, then place in a PCR instrument and perform the PCR reaction according to the program in Table 19.

[0105] Table 18 Reagents and dosages for enzymatic digestion

[0106] Table 19 PCR reaction procedure for enzyme digestion

[0107] After the PCR reaction program was completed, 110 μL of Vetaiseq DNA Clean Beads was added to the reaction tube and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then discarded. Next, 200 μL of an 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. The reaction tube was then incubated at room temperature for 8 min with the cap open. 20 μL of ddH2O was added to resuspend the dried magnetic beads. The tube was removed from the magnetic rack and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then transferred to a new tube, resulting in a 20 μL single-stranded circularized library. The library concentration was determined using a Qubit fluorometer.

[0108] 4. DNB preparation and sequencing DNB was prepared according to the instructions of the Vetaiseq DNBSEQ-G99RS high-throughput sequencing kit (PE150), calculating the required volume of single-stranded circularized library. DNB concentration was measured using a Qubit fluorometer, and sequencing was performed using a BGI Genomics DNBSEQ-G99RS gene sequencer. Each sample yielded 5 M Reads, and the sequencing time was 6–12 hours.

[0109] 4.1. Transgender The single-stranded library obtained in step 3.6 is mixed with other components according to the amounts specified in Table 20, vortexed and briefly centrifuged, and then placed in a PCR instrument to perform the PCR reaction according to the procedure in Table 21.

[0110] Table 20 Denaturing reagents and dosages

[0111] Table 21 PCR reaction procedure for denaturation

[0112] 4.2. DNB Preparation Prepare the reagents according to Table 22 and add 22 μL of the prepared reagents to the reaction tube after the reaction in step 4.1. Shake to mix and centrifuge briefly. Place the tube in a PCR instrument and perform the PCR reaction according to the procedure in Table 23. Once the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and gently pipette six times with a wide-mouth pipette tip. Store at 4°C before use.

[0113] Table 22 Reagents and dosages for DNB preparation

[0114] Table 23 PCR reaction procedure for DNB preparation

[0115] 4.3. DNB loading reagent preparation and sequencing Prepare the DNB loading system according to Table 24 and perform sequencing experiments according to the instructions for use of the BGI Genomics DNBSEQ-G99RS gene sequencer.

[0116] Table 24. Reagents and dosages used in the preparation of the DNB loading system.

[0117] 5. Bioinformatics Analysis (1) Data quality control: Use fastp / fastqc software to perform quality assessment and filtering on the raw sequencing data after the machine (remove low-quality reads, adapter sequences and short fragments). (2) Sequence alignment: The high-quality reads obtained after filtering were aligned to the FMDV reference genome using BWA-MEM; (3) Results statistics: Use samtools to calculate indicators such as comparison rate and coverage uniformity; (4) Coverage assessment: BamDst was used to calculate whole-genome coverage and average sequencing depth; (5) Mutation detection: Use bcftools or GATK to detect SNP and InDel mutations; (6) Evolutionary analysis: Multiple sequence alignment was performed using MAFFT, and maximum likelihood phylogenetic trees were constructed using IQ-TREE.

[0118] The results obtained using the sequencing methods described above are shown in Table 25. As can be seen from the results in Table 25, the amplification primer set of the present invention exhibits good specificity and sensitivity, achieving 99% genome coverage when sequencing nucleic acid samples with a nucleic acid concentration of 1 copy / μL or higher (the FMDV whole genome coverage diagram for nucleic acid sample ZKJY-E4 is shown in the figure below). Figure 2 As shown in the figure, the average sequencing depth is over 20,000X.

[0119] Table 25. Second-generation sequencing results

[0120] In Table 25: DF-1 is a negative control. After targeted amplification, the DNA concentration was found to be extremely low, indicating that targeted amplification failed and library construction and sequencing could not be performed.

[0121] Example 2 (Nanopore Sequencing) The experimental samples in this embodiment were: four nucleic acid samples ZKJY-E1, ZKJY-E2, ZKJY-E3, and ZKJY-E4 extracted from FMDV quality control materials and FMDV vaccine; ZKJY-E8, ZKJY-E9, ZKJY-E10, ZKJY-E11, and ZKJY-E12 were all obtained by diluting ZKJY-E4, with nucleic acid concentrations of 10000 copies / μL, 1000 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; DF-1 cell culture was used as a negative control to obtain sample DF-1; a total of 10 samples were used, with 3 replicates for each sample, for a total of 30 samples.

[0122] The samples ZKJY-E1, ZKJY-E2, and ZKJY-E3 were obtained as follows: 100 μL of FMDV quality control material was used to extract nucleic acids using the Vetaiseq DNA and RNA Co-extraction Kit (magnetic bead method) and the TGuide S16 fully automated nucleic acid extraction and purification instrument. The nucleic acids were then reconstituted using 90 μL of Elution Buffer to obtain a nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using the QuantStudio 1Plus real-time fluorescence quantitative PCR instrument, and the Ct value of the sample was obtained.

[0123] The ZKJY-E4 sample was obtained as follows: 100 μL of FMDV vaccine was used to extract nucleic acid using the Vetaiseq DNA and RNA Co-extraction Kit (magnetic bead method) and the TGuide S16 fully automated nucleic acid extraction and purification instrument. The nucleic acid was then reconstituted using 90 μL of ELution Buffer to obtain a nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using the QuantStudio 1Plus real-time fluorescence quantitative PCR instrument, and the Ct value of the sample was obtained.

[0124] The copy number of ZKJY-E4 samples was calculated using the Ct value of the ZKJY-E4 samples. Then, the ZKJY-E4 samples were diluted to 10000 copies / uL, 1000 copies / uL, 10 copies / uL, and 1 copy / uL to obtain nucleic acid samples ZKJY-E8, ZKJY-E9, ZKJY-E10, ZKJY-E11, and ZKJY-E12.

[0125] The nucleic acid purity of the above nucleic acid samples was determined using a NanoDrop micro spectrophotometer, and the nucleic acid concentration of the above nucleic acid samples was determined using a Qubit fluorometer.

[0126] The sequencing experiment process is as follows: First, the amplification products were obtained according to the methods described in steps 1-2 of Example 1. Then, the obtained amplification products were used to construct a library according to the instructions of the Vetaiseq Cyclone SEQ G100-ER 24-barcode rapid library construction kit, including end repair and dA addition, barcode and adapter ligation, and product purification. A final library recovery rate >10% was considered normal, and the library mass >250 ng, meeting the requirements for sequencing. Sequencing was performed using the Vetaiseq Cyclone SEQ G100-ER sequencing kit and the BGI Genomics Cyclone SEQ G100-ER sequencer, with a sequencing time of 0.5 h. The specific experimental procedure is as follows: 1.1. End-point repair and addition of dA The components in Table 26 are shaken to mix well and briefly centrifuged. The mixture is then placed in a PCR instrument and the PCR reaction is performed according to the procedure in Table 27. The volume of the amplified product (double-stranded DNA) is calculated based on the concentration of double-stranded DNA in the amplified product.

[0127] Table 26 Reagents and Doses for Terminal Repair and Addition of dA

[0128] Table 27 PCR reaction procedures for end repair and dA addition

[0129] 1.2. Barcode and connector connection Prepare reagents according to Table 28 using the product obtained in step 1.1 (terminal repaired dsDNA), vortex to mix and briefly centrifuge, then place in a PCR instrument to perform PCR reaction according to the program in Table 29.

[0130] Table 28 Reagents and Dosage for Barcode and Connector Connections

[0131] Table 29 PCR reaction procedures for barcodes and adapter ligation

[0132] After the PCR reaction program was completed, 24 μL of Vetaiseq DNA Clean Beads was added to the reaction tube and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then discarded. Next, 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. Another 200 μL of freshly prepared 80% ethanol aqueous solution was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. The magnetic beads were then dried at room temperature for 8 min. 17 μL of ddH2O was added to resuspend the dried magnetic beads. The magnetic rack was removed and incubated at room temperature for 5 min. The reaction tube was then transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then transferred to a new tube, resulting in 17 μL of supernatant. The DNA concentration was determined using the Vetaiseq 1×dsDNA HS Assay Kit.

[0133] 2. Sequencing Following the instructions for the Vetaiseq Cyclone SEQ G100-ER sequencing kit, the products obtained in step 1.2 were sequenced using the Vetaiseq Cyclone SEQ G100-ER sequencing kit and the BGI Genomics Cyclone SEQ-WT02 gene sequencer.

[0134] 3. Bioinformatics Analysis (1) Data quality control: Nanofilt was used to assess and filter the raw sequencing data after the sequencing machine (removing low-quality reads, adapter sequences and short fragments). (2) Sequence alignment: Minimap2 was used to align the filtered high-quality reads to the FMDV reference genome; (3) Results statistics: Use samtools to calculate indicators such as comparison rate and coverage uniformity; (4) Coverage assessment: BamDst was used to calculate whole-genome coverage and average sequencing depth; (5) Mutation detection: Use bcftools or GATK to detect SNP and InDel mutations; (6) Evolutionary analysis: Multiple sequence alignment was performed using MAFFT, and maximum likelihood phylogenetic trees were constructed using IQ-TREE.

[0135] The results obtained using the sequencing methods described above are shown in Table 30. As can be seen from the results in Table 30, the amplification primer set of this invention has good specificity and sensitivity, and the genome coverage is greater than 99% (the FMDV whole genome coverage diagram in nucleic acid sample ZKJY-E4 is shown in the figure below). Figure 3As shown in the figure, the lowest detection limit of 1 copy / μL can be achieved with an average sequencing depth of 1000X.

[0136] Table 30 Nanopore sequencing results

[0137] In Table 30: DF-1 is a negative control. After targeted amplification, the DNA concentration was found to be extremely low, indicating that targeted amplification failed and library construction and sequencing could not be performed.

[0138] Example 3 (Application of whole genome sequencing of O, A, Asia, and SAT1 serotypes of FMDV) The experimental samples in this embodiment are: four nucleic acid samples ZKJY-E4, ZKJY-E5, ZKJY-E6, and ZKJY-E7 extracted from the vaccine, with Ct values ​​of 13.6, 16.1, 14.2, and 17.7, respectively.

[0139] The samples ZKJY-E4, ZKJY-E5, ZKJY-E6, and ZKJY-E7 were obtained as follows: 100 μL of vaccine was taken and nucleic acid was extracted using the Vetaiseq DNA and RNA co-extraction kit and the TGuide S16 fully automated nucleic acid extraction and purification instrument. The nucleic acid was then reconstituted using 90 μL of Elution Buffer to obtain a nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was taken and qPCR was performed using the QuantStudio 1Plus real-time fluorescence quantitative PCR instrument, and the Ct value of each sample was obtained.

[0140] Sequencing experiments: (1) The amplification product was obtained according to steps 1 to 2 of Example 1.

[0141] (2) The amplification products obtained in step (1) were processed according to steps 3 to 5 of Example 1 to obtain the second-generation sequencing results, as shown in Table 31.

[0142] 3. The amplification products obtained in step 1 were processed according to steps 1 to 3 of Example 2 to obtain nanopore sequencing results, as shown in Table 31.

[0143] Table 31 Whole genome sequencing results of serotypes O, A, Asia, and SAT1 FMDV

[0144] In Table 31: the amount of data from second-generation sequencing is expressed as the number of sequences; the amount of data from nanopore sequencing is expressed as the number of bases.

[0145] As can be seen from the sequencing results in Table 31, the amplification primer set provided by this invention has good specificity and sensitivity, the sequencing method is compatible with second-generation sequencing platforms and nanopore sequencing platforms, and can realize whole-genome sequencing of serotypes O, A, Asia I, and SAT1 FMDV.

[0146] Based on the whole-genome phylogenetic tree, and using the sequencing results of this embodiment, the genotyping results of serotype A FMDV in nucleic acid sample ZKJY-E6 are as follows: Figure 4 As shown, the results of genotyping Asia I serotype FMDV in nucleic acid sample ZKJY-E7 are as follows: Figure 5 As shown, the results of genotyping FMDV serological type O in nucleic acid sample ZKJY-E5 are as follows: Figure 6 As shown, the results of genotyping SAT1 serotype FMDV in nucleic acid sample ZKJY-E4 are as follows: Figure 7 As shown.

[0147] Example 4 (Application of whole genome sequencing of FMDV serotypes C, SAT2, and SAT3) Given the scarcity of clinical samples and quality control materials for FMDV serotypes C, SAT2, and SAT3 both domestically and internationally (serotype C has not been detected in natural circulation globally since 2004, and serotypes SAT2 and SAT3 are mainly confined to sub-Saharan Africa), this invention employs a combined strategy of "primer matching analysis + simulated sequencing verification" to systematically verify the applicability of the primer set of this invention to serotypes C, SAT2, and SAT3 from a bioinformatics perspective, and compares it with the experimental results of serotypes O, A, Asia I, and SAT1. The specific process is as follows: 1. Construction of the reference sequence set Download the complete genome sequences of each serotype of FMDV up to June 2026 from the GenBank database to construct a reference sequence set. For specific download instructions, log in to: https: / / www.ncbi.nlm.nih.gov / labs / virus / vssi / # / virus? and download the genome reference sequences for serotypes O, A, C, Asia I, SAT1, SAT2, and SAT3 of FMDV respectively. Table 32 Genomic reference sequences of different serotypes of FMDV

[0148] In Table 32, a indicates that the complete genome was obtained from the NCBI public database; b indicates that the reference strain was the reference strain used when designing the primers. 2. Primer-serum type matching analysis The 11 primer pairs (22 sequences) in Table 4 were sequentially aligned to the complete genome sequences of each serotype of FMDV in the aforementioned reference sequence set. The matching was assessed using BLASTn and Primer-BLAST tools. Assessment metrics included: (1) 3' end matching degree: the matching degree of the last 5 bases at the 3' end of the primer with the reference sequence (complete matching or only 1 mismatch is considered as qualified); (2) Total number of mismatches: The total number of mismatches between the full-length primer sequence and the corresponding regions of the reference sequence (≤5 mismatches are considered acceptable); (3) Estimated Tm value change: The effect of mispaired primer annealing temperature (ΔTm ≤ 10℃ is considered acceptable).

[0149] Primer-serum type matching heatmap as shown Figure 8 As shown in Table 33, the results of primer-serum type matching analysis are summarized below.

[0150] Table 33 Summary of Primer-Serotype Matching Results

[0151] The results in Table 33 show that: (1) The overall primer matching rate for serotypes O, A, Asia I, and SAT1 ranged from 83% to 98.4%, with some primers having a lower matching rate, but this did not affect the amplification of the whole genome sequence. This is consistent with the experimental verification results of Examples 1 to 3, proving that the matching degree analysis method is reliable.

[0152] (2) The primer matching rates for serotypes C, SAT2 and SAT3 were 100%, 98.7% and 96.4% respectively, all of which were higher than 95%.

[0153] 3. Simulated sequencing data analysis 3.1 Simulating read generation One representative reference genome each from C, SAT2, and SAT3 was selected (see Table 32). Based on the amplifiable regions identified through primer matching analysis, simulated sequencing reads were generated using the ART tool (next-generation sequencing simulation) and the NanoSim tool (nanopore sequencing simulation), respectively. (1) Second generation sequencing simulation parameters: MGISEQ platform, PE150, simulated sequencing depth 1000X, base quality distribution refers to actual sequencing data; (2) Nanopore sequencing simulation parameters: Nanopore platform, average read length 1200 bp, simulated sequencing depth 1000X, error model referenced Nanopore R10 chemical system.

[0154] 3.2 Bioinformatics Analysis Workflow The simulated reads were processed according to the standard bioinformatics analysis procedures of Example 1 (next-generation sequencing) and Example 2 (nanopore sequencing), and the simulation verification results are shown in Table 34.

[0155] Table 34 Whole-genome sequencing results of different serotypes of FMDV

[0156] In Table 34, the measured data are from Example 3; the simulated data are from the simulation experiment in this example. The results in Table 34 show that: (1) The simulation analysis results show that the genome coverage of serotypes C, SAT2, and SAT3 FMDV reached (99.89% / 99.98%), (99.69% / 99.77%), and (99.83% / 99.92%), respectively, which are at the same level as the measured results of serotypes O, A, Asia I, and SAT1 FMDV (>99%). (2) The average sequencing depth reached over 1000X, meeting the requirements for serological typing and variant analysis; (3) The nanopore sequencing coverage maps of serotypes C, SAT2, and SAT3 FMDV are shown below. Figure 9 , Figure 10 , Figure 11 As shown; Figures 9-11 The results show that the coverage of all regions of the whole genome is uniform, with no obvious gaps, confirming that the primer set of the present invention is also effective against C, SAT2, and SAT3 serotypes of FMDV.

[0157] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A primer set for amplifying the entire genome of foot-and-mouth disease virus, characterized in that, The amplification primer set includes 11 pairs of primers, the sequences of the upstream and downstream primers of the 11 pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO.

22.

2. An amplification reagent targeting the entire genome of foot-and-mouth disease virus, characterized in that, The amplification reagent includes the amplification primer set as described in claim 1.

3. An amplification kit for the whole genome of foot-and-mouth disease virus, characterized in that, The amplification kit includes the amplification primer set as described in claim 1 or the amplification reagent as described in claim 2.

4. A method for amplifying the entire genome of foot-and-mouth disease virus for non-disease diagnosis and treatment purposes, characterized in that, The amplification method includes amplification using the amplification primer set of claim 1, the amplification reagent of claim 2, or the amplification kit of claim 3.

5. The application of the amplification method according to claim 4 in the identification of foot-and-mouth disease virus for non-disease diagnosis and treatment purposes.

6. A sequencing method for the entire genome of foot-and-mouth disease virus for non-disease diagnosis and treatment purposes, characterized in that, The sequencing method includes performing multiplex targeted amplification using the amplification primer set of claim 1, the amplification reagent of claim 2, or the amplification kit of claim 3.

7. The sequencing method according to claim 6, characterized in that, The sequencing method includes the following steps: (1) Collect samples and extract nucleic acids from the samples; (2) The nucleic acid extracted in step (1) is reverse transcribed to obtain cDNA; (3) Use the amplification primer set of claim 1, the amplification reagent of claim 2, or the amplification kit of claim 3 to perform multiple targeted amplification of the cDNA obtained by reverse transcription in step (2); (4) Purify the nucleic acid of the amplification product obtained in step (3); (5) Constructing sequencing libraries and performing sequencing; (6) Bioinformatics analysis.

8. The sequencing method according to claim 7, characterized in that, The foot-and-mouth disease virus includes one or more serotypes of foot-and-mouth disease virus, namely O, A, C, Asia I, SAT1, SAT2 and SAT3. And / or, the sample in step (1) includes one or more of the following: nasal and oral swabs, throat swabs, vesicular fluid, and tissue homogenate. And / or, the method for nucleic acid extraction in step (1) includes extraction using a nucleic acid extraction kit; And / or, the method of reverse transcription in step (2) includes cDNA synthesis using a reverse transcription kit; And / or, when performing multiplex targeted amplification in step (3), the 11 pairs of primers in the amplification primer set described in claim 1 are divided into two groups and subjected to multiplex targeted amplification with the cDNA obtained by reverse transcription in step (2), wherein primer set 1 consists of primers with the sequences shown in SEQ ID NO. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, and primer set 2 consists of primers with the sequences shown in SEQ ID NO. 3, 4, 7, 8, 11, 12, 15, 16, 19, 20; And / or, the length of the amplification product obtained by multiple targeted amplification in step (3) is 1000-2000 bp; And / or, the procedure for multiple targeted amplification in step (3) is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ denaturation for 30s, 72℃ denaturation for 90s, for a total of 25 cycles; 72℃ extension for 5min; And / or, the reaction reagents for the multiplex targeted amplification in step (3) include Pathogen DNA Multiplex PCRMix, ddH2O, the amplification primer set according to claim 1, and the cDNA obtained in step (2); And / or, the nucleic acid purification method in step (4) includes purification using purification magnetic beads; And / or, step (5) of constructing the sequencing library is performed on a next-generation sequencing platform or a nanopore sequencing platform; And / or, the bioinformatics analysis in step (6) includes data quality control, sequence alignment, result statistics, coverage assessment, variant detection and evolutionary analysis.

9. The sequencing method according to claim 8, characterized in that, The construction of the sequencing library in step (5) is performed on a second-generation sequencing platform. The construction of the sequencing library specifically includes the following steps: fragment fragmentation, end repair and dA addition, adapter ligation, library amplification, denaturation, single-strand circularization, enzyme digestion, and DNB preparation. Alternatively, step (5) of constructing the sequencing library is performed on a nanopore sequencing platform. The construction of the sequencing library specifically includes the following steps: end repair and addition of dA, barcode and adapter ligation.

10. The use of the sequencing method according to any one of claims 6 to 9 in serotyping of foot-and-mouth disease virus for non-disease diagnosis and treatment purposes.

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