Primer composition and method for whole genome sequencing of American type porcine reproductive and respiratory syndrome virus and application of primer composition and method

Abstract

The invention relates to a primer composition and a method for whole genome sequencing of American type porcine reproductive and respiratory syndrome virus and application of the primer composition and the method. The sequences of the primer composition are as shown in SEQ ID No. 1 to SEQ ID No. 35. According to the sequencing method, RNA (Ribonucleic Acid) of a sample to be detected is taken as a template to perform reverse transcription reaction, the primer combination is adopted to perform PCR (Polymerase Chain Reaction) amplification on the whole genome of the American type porcine reproductive and respiratory syndrome virus, and an amplification product is subjected to three-generation sequencing library building and sequencing to perform species identification and genetic typing. The full-length PCR detection method is high in specificity and sensitivity, the efficiency of detecting the whole genome of the virus is greatly improved, rapid and accurate detection of the American type porcine reproductive and respiratory syndrome virus is achieved, accurate typing and evolutionary analysis can be achieved by utilizing the assembled whole genome, and the method is suitable for large-scale popularization and application. Traceability of the reproductive and respiratory syndrome virus is realized, and the method is of great significance to early screening, prevention and control of the American porcine reproductive and respiratory syndrome virus.

Description

Technical Field

[0001] The present invention relates to the technical field of animal virus detection, and in particular to a primer composition, method and application thereof for whole genome sequencing of American porcine reproductive and respiratory syndrome virus. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae and is a positive-strand RNA virus. The virus appears in both North America and Europe and has a significant economic impact on the pig industry worldwide, including China. PRRSV can be divided into two genotypes, the American type and the European type, both of which can cause abortion, stillbirth and respiratory diseases in sows. The American type is the main genotype in China. The American type of PRRSV is divided into 9 lineages (1-9), and each lineage can be further divided into several sublineages. In 2006, a highly pathogenic PRRSV (HP-PRRSV) (belonging to Lineage 8) with an extremely high mortality rate appeared in Jiangxi Province, China, and it became prevalent in many provinces in a short period of time, which once brought a devastating blow to my country's pig industry. In 2012, Zhou Feng et al. isolated strains in my country that were highly homologous to NADC30 and had the same deletion pattern in the NSP2 region. Chinese scholars called strains with such deletion characteristics NADC30-like strains (belonging to Lineage 1, Sublineage 1.8). In 2017, the existence of NADC34 strains was first detected in Liaoning Province, my country. Because they were similar to the IA / 2014 / NADC34 strain in the United States and had a continuous deletion of 100 amino acids in the NSP2 region, this type of strain was called NADC34-like strain (belonging to Lineage 1, Sublineage 1.5). Data show that in recent years, the detection rate of NADC34-like strains in my country has continued to increase, from less than 3% in 2017-2019 to 11.5% in 2020, and as high as 28.6% in 2021. In 2022, the detection rate remains high. At this stage, it has become the main prevalent strain in my country together with NADC30-like strains (35.4%) and HP-PRRSV (31.2%). It has been reported that since October 2020, a new variant of PRRSV 1-4-4 Lineage 1C has broken out in many places in the United States. The transmission ability and pathogenicity of this strain are stronger than previous American strains. There are currently precedents of multiple American PRRSV strains being introduced into my country and becoming widely prevalent in the country. The genome of the PRRSV strain is very prone to mutation, and different subtypes of PRRSV are prone to recombination, and new variant recombinant strains are derived. The prevalence of these variants has increased the diversity of PRRSV in my country. Genetic recombination is an important means of PRRSV evolution. Recombination can occur between different lineages of PRRSV strains and give rise to new variant recombinant strains.For example, the FJLIUY-2017 strain isolated in Fujian Province in 2017 was derived from the recombination of multiple lineages of Lineage 1, Lineage 3, Lineage 5 and Lineage 8. According to data from the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, during the period of 2014-2018, the United States was mainly recombined with Lineage 1+Lineage 5, and China was mainly recombined with Lineage 1+Lineage 8. In recent years, the recombination frequency of Lineage 1 lineage in my country is higher than that of Lineage 8 lineage. High mutation and easy recombination are the significant characteristics of American porcine reproductive and respiratory syndrome virus. The fluorescent quantitative PCR scheme on the market only detects certain specific gene conservative regions, and there are cases where some lineage strains are missed. At present, the commonly used methods include conventional virus isolation and serum detection, which are often time-consuming and cumbersome. It is urgent to establish a fast, highly sensitive, comprehensive analysis and easy-to-operate method to achieve early diagnosis of the disease.

[0003] At present, the main methods for whole genome sequencing include: (1) Traditional Sanger sequencing, which has the advantages of long sequencing read length and high accuracy, and is suitable for detailed analysis and verification of viral genomes; its disadvantages are low throughput, high cost, and slow speed, and is not suitable for large-scale viral whole genome sequencing; (2) High-throughput sequencing technology: also known as new generation sequencing technology, mainly including second-generation sequencing technology (such as Illumina sequencing) and third-generation sequencing technology (such as PacBio sequencing, Nanopore sequencing), etc. The advantages of the Illumina sequencing method are high throughput, low cost, and good accuracy. It is suitable for large-scale viral whole genome sequencing and population genetics research. The disadvantage is that the read length is relatively short, and for longer viral genomes, splicing and assembly are required; the advantage of the PacBio sequencing method is that the complete viral genome can be directly measured without complex splicing and assembly, and it has advantages in detecting structural variations and repetitive sequences of the viral genome. The disadvantages are relatively low throughput, high cost, and a relatively high sequencing error rate; the advantages of the Nanopore sequencing method are long read length, fast sequencing speed, and portable instruments. It can be rapidly sequenced on-site or in the field, which is of great significance for real-time monitoring of virus mutation and spread. The disadvantages are relatively low throughput and relatively high error rate, and the data processing method needs to be further optimized to improve accuracy.

[0004] However, existing sequencing methods rarely involve the sequencing of the whole genome sequence of American porcine reproductive and respiratory syndrome virus, and there is no design of shingled PCR primer sequences related to the whole genome sequencing of American porcine reproductive and respiratory syndrome virus. Therefore, the development of a third-generation whole genome sequencing method for American porcine reproductive and respiratory syndrome virus is of great significance for the diagnosis of the disease and the formulation of its prevention and control strategies. Summary of the invention

[0005] The purpose of the present invention is to address at least one of the shortcomings of the prior art and to provide a primer composition, method and application thereof for whole genome sequencing of the American porcine reproductive and respiratory syndrome virus. The above-mentioned primer composition and sequencing method can realize simultaneous amplification of the full-length genome of the American porcine reproductive and respiratory syndrome virus in one reaction system without the need to remove host nucleic acid, thereby saving experimental operation time.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a primer composition for full-length amplification of the genome of American porcine reproductive and respiratory syndrome virus, which comprises the sequences shown in SEQ ID No. 1 to SEQ ID No. 35. The primer composition is a shingled PCR primer.

[0008] Furthermore, the primer composition is divided into a primer group A and a primer group B, the primer group A consists of the sequences shown in SEQ ID No.1 to SEQ ID No.19, and the primer group B consists of the sequences shown in SEQ ID No.20 to SEQ ID No.35.

[0009] The specific information of the above primer sequences is shown in the following table:

[0010] Table 1 - Primers for PCR

[0011]

[0012]

[0013] The second aspect of the present invention is to provide a kit for the primer combination described in any one of the first aspects, which also includes a reverse transcription reagent, an amplification reaction reagent, and a product purification reagent.

[0014] The reverse transcription reagent may be LunaScript RT SuperMix (5X) reverse transcription mixture (NEB, E3010S, containing random hexamer and poly-dT primers), and the amplification reaction reagent may be KOD One TM PCR Master Mix (KMM-101), the above product purification reagents include magnetic beads.

[0015] The third aspect of the present invention is to provide a method for sequencing the whole genome of the American porcine reproductive and respiratory syndrome virus, which comprises the following steps:

[0016] Step S1, collecting a sample, using the viral RNA extracted from the sample as a template, and performing reverse transcription using a reverse transcription reagent to obtain cDNA;

[0017] Step S2, using the cDNA obtained in step S1 as a template, and using an amplification reaction reagent and any primer combination described in the first aspect to perform a PCR reaction;

[0018] Step S3: After the PCR reaction is completed, a product purification reagent is used to remove excess impurities in the reaction product obtained in step S2;

[0019] Step S4: construct a library and perform sequence determination on the purified product obtained in step S3.

[0020] Furthermore, in step S1, the sample includes a body fluid sample, a tissue sample, and a feces sample.

[0021] Furthermore, in step S1, the reverse transcription reagent is RT SuperMix (5x), the reverse transcription reaction system is 8 μL viral RNA, 2 μL RT SuperMix (5x), and the reverse transcription reaction procedure is: 25°C, 2 min; 55°C, 10 min; 95°C, 1 min.

[0022] Further, in step S2, the PCR reaction includes reaction system A and reaction system B, wherein reaction system A is 8 μL of group A primers, 4.5 μL of cDNA, and 12.5 μL of 2x PCR Master Mix; reaction system B is 8 μL of group B primers, 4.5 μL of cDNA, and 12.5 μL of 2x PCR Master Mix; wherein the concentration of each group of primers is 10 μM; the reaction procedures of each reaction system are: 94°C for 2 min; 98°C for 10 s, 58°C for 5 s, 68°C for 1 min, 35 cycles; 68°C for 5 min.

[0023] Furthermore, in step S3, the product purification reagent includes magnetic beads, 18 μL of magnetic beads are added to reaction product A and reaction product B respectively for purification, and 20 μL of nuclease-free water is used to collect purified product A and purified product B.

[0024] Furthermore, in step S3, the purified product meets the initial quality and purity requirements for library construction for nanopore sequencing.

[0025] Furthermore, in step S4, the library construction and sequence determination specifically include:

[0026] Step S41, on a nanopore sequencing platform, using the SQK-LSK114.24 kit to construct a library;

[0027] Step S42, loading the library constructed in step S41 onto the nanopore sequencing chip, setting the reaction program, and performing sequencing;

[0028] Step S43: analyzing the sequencing data obtained in step S42.

[0029] Furthermore, in step S41, the library construction step includes:

[0030] a) End repair: NEB repair enzyme was used, and the repair reaction system was 0.3 μL end repair enzyme, 0.7 μL end repair enzyme buffer, 30-100 ng purified product A, 30-100 ng purified product B, and nuclease-free water was added to 7 μL; the reaction procedure was: 20°C for 2 min, 65°C for 5 min;

[0031] b) Tag connection: Add a specific Barcode tag sequence to each sample, and the reaction system is: 1μL Barcode tag, 5μL Barcode premix, 4μL end repair product; after incubation at room temperature for 10 minutes, add 7μL magnetic beads for purification, and use 6μL nuclease-free water to collect the mixed product connected to the Barcode;

[0032] c) Sequencing adapter ligation: The reaction system is 2 μL sequencing adapter, 2 μL DNA ligase, 4 μL NEB buffer, and 12 μL mixed product of Barcode ligation; wherein, the amount of mixed product of Barcode ligation in the reaction system is 250 ng, and incubation is carried out at room temperature for 10 min.

[0033] Furthermore, in step S42, the nanopore sequencing chip is FLO-MIN114 R10.4.1, and the sequencing software is GridION.

[0034] Furthermore, in step S43, the analysis platform is the BoChuangyi porcine reproductive and respiratory syndrome (PRRSV) virus bioinformatics analysis cloud platform.

[0035] The fourth aspect of the present invention is to provide an application of any primer-probe combination described in the first aspect, or any kit described in the second aspect, or any sequencing method described in the third aspect in the whole genome sequencing of American porcine reproductive and respiratory syndrome virus.

[0036] Furthermore, the application includes early identification and genotyping of the American porcine reproductive and respiratory syndrome virus. Virus identification and typing can provide a certain basis for understanding viral genetic variation, studying viral pathogenicity, vaccine development, and formulation of disease prevention and control strategies.

[0037] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0038] (1) The primer combination of the present invention can effectively enrich and specifically obtain the whole genome sequence of the American type porcine reproductive and respiratory syndrome virus, and the target sequence matched accounts for 66.6% of the offline data. In addition, the low-concentration sample with a Ct value of 30 detected by fluorescent quantitative PCR can also obtain an average depth of 1828× and a coverage of 99.853%;

[0039] (2) The whole genome amplification primer composition and method provided by the present invention have low cost, simple operation, accurate detection results, and are conducive to the early identification and typing of American porcine reproductive and respiratory syndrome virus, thereby providing a basis for the formulation of disease prevention and control measures and the development of related vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0041] Figure 1 A flowchart of a sequencing method in one embodiment of the present invention;

[0042] Figure 2 Schematic diagram of the design of shingled PCR primers in one embodiment of the present invention;

[0043] Figure 3 It is a full-length genome amplification coverage and depth map in one embodiment of the present invention;

[0044] Figure 4 This is a full-length genome amplification coverage map in one embodiment of the present invention;

[0045] Figure 5 This is an electrophoresis diagram of PCR products in one embodiment of the present invention;

[0046] Figure 6 A Sankey diagram for species identification in one embodiment of the present invention;

[0047] Figure 7 This is a species classification diagram in one embodiment of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0049] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0050] Example 1 - Design of amplification primers for third-generation sequencing of the entire genome of American porcine reproductive and respiratory syndrome virus

[0051] This example uses shingled PCR amplification technology (such as Figure 2 The purpose is to enrich the PRRSV virus in the sample and capture sequence fragments. There are overlapping regions between these amplified fragments, covering the entire length of the genome.

[0052] According to the conserved region sequences of the genomes of various subtypes of porcine reproductive and respiratory syndrome virus in the American type published by the National Center for Biotechnology Information (NCBI), large fragment deletions and hypervariable regions were spanned and degenerate bases were introduced to cover more subtypes. 35 primer sequences (SEQ ID No. 1 to SEQ ID No. 35) were designed using molecular biology software such as BioEdit. The specific sequence information is shown in Table 1 above.

[0053] The above 35 primer sequences can be divided into two groups, among which SEQ ID No.1 to SEQ ID No.19 are PoolA, and SEQ ID No.20 to SEQ ID No.35 are PoolB. When designing the primer set, the principle of independent amplification of two sets of primers (divided into PoolA and PoolB) is followed. While amplifying the whole genome, it can also avoid the generation of a large number of short fragments by adjacent primers to cause amplification preference. Except for the A3 primer pair (amplification product is 2104bp) and the A2 primer pair (amplification product is 1844bp) in the primer pair PoolA, the purpose is to cross the deletion of large fragments. The fragment sizes of other primers are all around 1000bp to ensure the consistency of amplification efficiency as much as possible; in addition, an A2R ALT is added to the downstream primer of the A2 primer to make up for the defect of Gap when A2F is used alone, so that the amplified genome sequence is more complete.

[0054] Example 2-Establishment of a third-generation sequencing method for the whole genome of American porcine reproductive and respiratory syndrome virus

[0055] In this example, based on the shingled PCR primers designed in Example 1, a third-generation sequencing method for the whole genome of the American porcine reproductive and respiratory syndrome virus was established. The process is as follows: Figure 1 As shown, the specific steps include:

[0056] (1) Experimental materials;

[0057] Viral RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP315-R), Reverse transcription kit (NEB, E3010S), PCR amplification reagent kod oneTM PCR Master Mix (TOYOBO, KMM-101), amplification-free barcoding kit-24V14 (ONT, SQK-NBD114.24).

[0058] (2) Sequencing method;

[0059] 1) Preparation of viral RNA

[0060] The viral RNA in the sample was extracted using a viral RNA extraction kit, and the prepared viral RNA was tested by qPCR (Beijing Mingrida Technology Development Co., Ltd., 0001007014). The recommended Ct value was less than 30. The reaction system for qPCR detection was 25 μL. 20 μL of fluorescent PCR reaction solution was added to the PCR tube, and 5 μL of sample, negative control, and positive control were added. The reaction conditions were as follows:

[0061]

[0062] 2) Reverse transcription

[0063] The reverse transcription reaction system is 10 μL, of which 8 μL is viral RNA and 2 μL is RT SuperMix (5x) (NEB, E3010S, containing random hexamer and poly-dT primer). The system is mixed by flicking with fingers and centrifuged briefly. The reverse transcription reaction conditions are as follows: After reverse transcription, the viral cDNA of the sample is obtained.

[0064]

[0065] 3) PCR amplification

[0066] Using the reverse transcribed viral cDNA as a template, two reaction systems were prepared independently according to Pool A (containing only A primer pair, SEQ ID No. 1 to SEQ ID No. 19) and Pool B (containing only B primer pair, SEQ ID No. 20 to SEQ ID No. 35) to prevent cross-reaction between primers and cause non-specific amplification. Among them, 2x PCR Master Mix was KOD One TM Components of the PCR Master Mix (KMM-101) kit.

[0067] Reagents Pool A (μL) Pool B (μL) 2x PCR Master Mix 12.5 12.5 cDNA 4.5 4.5 Pool A Primers (10 μM) 8 - Pool B Primers (10 μM) - 8 Total 25 25

[0068] PCR reaction conditions:

[0069]

[0070] 4) PCR product purification

[0071] The magnetic beads were mixed by oscillation, and 18 μL of magnetic beads were added to the two PCR reaction tubes in 3) for purification. Two PCR amplification products were collected with 20 μL of Nuclease-free water respectively, and the purified products were quantified with Qubit.

[0072] 5) Library construction and sequencing

[0073] a) End repair: Take 100 ng (if less than 100 ng, 30-100 ng can be input) of the purified product in 4) and perform end repair operation. The reaction system is:

[0074]

[0075]

[0076] The reaction conditions are:

[0077]

[0078] b) Label connection: In order to distinguish different samples in a reaction system, a specific Barcode label sequence (ONT, SQK-NBD114.24) can be added to each sample. The reaction system is:

[0079] Reagents Volume (μL) Barcode Tags 1 Barcode Master Mix 5 a) Mid-term and final repair products 4 Total 10

[0080] After incubation at room temperature for 10 min, 7 μL of magnetic beads were added for purification, and the Barcode-connected products were collected with 6 μL of Nuclease-free water. After purification, the products were quantified using Qubit.

[0081] c) Adapter connection: Sequencing adapters with motor proteins (ONT, SQK-NBD114.24) can connect to the barcode sequence and pull the nucleic acid molecules into the protein nanopore to be recognized as base signals. Specifically, the purified products in b) need to be used for adapter connection. The purified products of barcode connection of each sample are mixed in equal amounts, a total of 250ng, and the adapter connection reaction is carried out. The reaction system is:

[0082] Reagents Volume (μL) Native Adapter(NA) 2 Quick T4 DNA ligase 2 NEB next quick ligation reaction buffer(5X) 4 Pooled library DNA 250ng 12 Total 20

[0083] After incubation at room temperature for 10 min, the library was prepared according to the official (ONT, SQK-NBD114.24) standard protocol loading process, loaded into the chip (ONT, FLO-MIN114 R10.4.1), and run on the GridION (ONT) sequencer to obtain sequencing data.

[0084] In this embodiment, the protocol of the official (ONT, SQK-NBD114.24) kit is appropriately adjusted. Specifically, the reaction system in the PCR product end repair process is optimized from the original 15 μL to 7 μL, and the magnetic bead purification of each sample is omitted, and the next step of Barcode connection is directly performed to shorten the process time; the reaction system in the label connection process is optimized from the original 20 μL to 10 μL; the reaction system in the connector connection process is optimized from the original 50 μL to 20 μL to save costs.

[0085] 6) Data analysis: The data generated by sequencing can be directly analyzed on the Porcine Reproductive and Respiratory Syndrome (PRRSV) Virus Bioinformatics Analysis Cloud Platform. The specific analysis includes: a) Quality control of the original data, filtering out sequences with a q value lower than 9 and a length less than 200bp; b) Comparing the quality-controlled data to the NCBI virus library, identifying the species, and obtaining the species identification Sankey diagram; c) Selecting the reference sequence, performing reference assembly, and calculating the depth of the reads after quality control; d) Comparing the assembled genes to the blue ear virus (porcine reproductive and respiratory syndrome virus) database, drawing an evolutionary tree, and performing typing analysis.

[0086] Example 3 - Practical application of sequencing method

[0087] In this example, the sequencing method constructed in Example 2 was used to perform full-length genome amplification, sequencing and identification of a case of American porcine reproductive and respiratory syndrome virus in a pig farm.

[0088] In June 2024, piglets in a pig farm developed respiratory symptoms such as coughing and wheezing, depression, loss of appetite, fever, conjunctival redness, increased tearing, and redness of the ears, skin, and groin at the beginning of the disease. As the disease progressed, the skin turned blue-purple. Some pigs had hindquarters weakness, inability to stand, or ataxia. After preliminary clinical diagnosis, porcine reproductive and respiratory syndrome was suspected. The inguinal lymph nodes of dying pigs were collected and placed in sterile containers, immediately refrigerated and sent to the laboratory.

[0089] (1) Sample pretreatment;

[0090] Use sterile scissors and forceps to cut 1.0 g of inguinal lymph node sample and grind it thoroughly in a mortar. Then add 1 mL of PBS to mix well. Then transfer the tissue suspension into a 1.5 mL sterile centrifuge tube and centrifuge at 3000 r / min for 10 min. Transfer the supernatant into another sterile centrifuge tube and number it for later use.

[0091] (2) Extraction of viral RNA;

[0092] The viral RNA in the sample was extracted using a viral RNA extraction kit, and the prepared viral RNA was subjected to qPCR detection (Beijing Mingrida Technology Development Co., Ltd., 0001007014), with a Ct value of 20.43. The qPCR detection system is shown in Example 2.

[0093] (3) The extracted viral RNA was sequenced and analyzed using the whole genome third-generation sequencing method of Example 2.

[0094] The sequencing results showed that the above sequencing method can amplify, identify and type the full-length genome of the American type of porcine reproductive and respiratory syndrome virus. Figure 3After the assembled genome was aligned with the reference genome, the calculated coverage was as high as 99.853% ( Figure 4 ), indicating that the assembly result is good. The electrophoresis diagram of the PCR amplification product is shown in Figure 5 The size of the amplification product of each pair of primers conforms to the theoretical size of the primer design product; and the sample 1 can be identified in a relatively short time (e.g. Figure 5 ) and classification (such as Figure 6 ), most of the sequences were compared to the database and identified as Betaarterivirus suid2, the North American type of PRRSV. In the typing analysis, it was considered to be closely related to the L1C.3 type (belonging to Lineage 1) variant strain.

[0095] As can be seen from the above embodiments, the present invention is based on the PCR technology of the shingled primer combination, and a total of 35 primers are designed. The RNA of the sample to be tested is used as a template for reverse transcription reaction, and the primer combination shown in SEQ ID No.1 to SEQ ID No.35 is used to perform PCR amplification on the full-length genome of the American porcine reproductive and respiratory syndrome virus, and then the amplified product is subjected to three-generation sequencing library construction and sequencing, and species identification and typing are performed, which can achieve effective amplification of the full-length genome of the American porcine reproductive and respiratory syndrome virus, and effectively enrich the American porcine reproductive and respiratory syndrome virus while reducing the interference of host nucleic acid, reducing the cost of sequencing, and shortening the sequencing time. The full-length PCR detection of the present invention has strong specificity and high sensitivity, greatly improving the efficiency of measuring the whole genome of this type of virus, not only realizing the rapid and accurate detection of the American porcine reproductive and respiratory syndrome virus, but also being able to use the assembled whole genome to achieve accurate typing and evolutionary analysis, and realizing the tracing and tracing of the reproductive and respiratory syndrome virus, which is of great significance to the early screening and prevention and control of the American porcine reproductive and respiratory syndrome virus.

[0096] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer composition, characterized in that The primer combination is used for full-length amplification of the American porcine reproductive and respiratory syndrome virus genome, and includes sequences shown as SEQ ID No.1 to SEQ ID No.

35.

2. The primer composition according to claim 1, characterized in that The primer composition is divided into a primer group A and a primer group B. The primer group A consists of sequences shown in SEQ ID No.1 to SEQ ID No.19, and the primer group B consists of sequences shown in SEQ ID No.20 to SEQ ID No.

35.

3. A kit containing the primer combination according to claim 1 or 2, characterized in that: The kit also includes a reverse transcription reagent, an amplification reaction reagent, and a product purification reagent.

4. A method for sequencing the whole genome of the American porcine reproductive and respiratory syndrome virus, characterized in that: The steps include: Step S1, collecting a sample, using the viral RNA extracted from the sample as a template, and performing reverse transcription using a reverse transcription reagent to obtain cDNA; Step S2, using the cDNA obtained in step S1 as a template, and using an amplification reaction reagent and the primer combination as claimed in claim 1 or 2 to perform a PCR reaction; Step S3: After the PCR reaction is completed, a product purification reagent is used to remove excess impurities in the reaction product obtained in step S2; Step S4: construct a library and perform sequence determination on the purified product obtained in step S3.

5. The sequencing method according to claim 4, characterized in that In step S1, the sample includes a body fluid sample, a tissue sample, and a fecal sample; the reverse transcription reagent is RTSuperMix (5x), the reverse transcription reaction system is 8 μL viral RNA, 2 μL RT SuperMix (5x), and the reverse transcription reaction procedure is: 25°C, 2 min; 55°C, 10 min; 95°C, 1 min; and / or, In step S2, the PCR reaction includes reaction system A and reaction system B, wherein reaction system A is 8 μL of group A primers, 4.5 μL of cDNA, and 12.5 μL of 2x PCR Master Mix; reaction system B is 8 μL of group B primers, 4.5 μL of cDNA, and 12.5 μL of 2x PCR Master Mix; wherein the concentration of each group of primers is 10 μM; the reaction procedures of each reaction system are: 94°C for 2 min; 98°C for 10 s, 58°C for 5 s, 68°C for 1 min, 35 cycles; 68°C for 5 min; and / or, In step S3, the product purification reagent includes magnetic beads, 18 μL of magnetic beads are added to reaction product A and reaction product B respectively for purification, and 20 μL of nuclease-free water is used to collect purified product A and purified product B.

6. The sequencing method according to claim 4 or 5, characterized in that In step S3, the purified product meets the initial quality and purity requirements for library construction for nanopore sequencing; in step S4, the library construction and sequence determination specifically include: Step S41, on a nanopore sequencing platform, using the SQK-LSK114.24 kit to construct a library; Step S42, loading the library constructed in step S41 onto the nanopore sequencing chip, setting the reaction program, and performing sequencing; Step S43: analyzing the sequencing data obtained in step S42.

7. The sequencing method according to claim 6, characterized in that: In step S41, the library construction step includes: a) End repair: NEB repair enzyme was used, and the repair reaction system was 0.3 μL end repair enzyme, 0.7 μL end repair enzyme buffer, 30-100 ng purified product A, 30-100 ng purified product B, and nuclease-free water was added to 7 μL; the reaction procedure was: 20°C for 2 min, 65°C for 5 min; b) Tag connection: Add a specific Barcode tag sequence to each sample, and the reaction system is: 1μL Barcode tag, 5μL Barcode premix, 4μL end repair product; after incubation at room temperature for 10 minutes, add 7μL magnetic beads for purification, and use 6μL nuclease-free water to collect the mixed product connected to the Barcode; c) Sequencing adapter ligation: The reaction system is 2 μL sequencing adapter, 2 μL DNA ligase, 4 μL NEB buffer, and 12 μL mixed product of Barcode ligation; wherein, the amount of mixed product of Barcode ligation in the reaction system is 250 ng, and incubation is carried out at room temperature for 10 min.

8. The sequencing method according to claim 6, characterized in that In step S42, the nanopore sequencing chip is FLO-MIN114R10.4.1, and the sequencing software is GridION; and / or, in step S43, the analysis platform is the BoChuangyi porcine reproductive and respiratory syndrome (PRRSV) virus bioinformatics analysis cloud platform.

9. Use of the primer combination as described in any one of claims 1 to 2, or the kit as described in claim 3, or the sequencing method as described in any one of claims 4 to 8 in whole genome sequencing of American porcine reproductive and respiratory syndrome virus.

10. The use according to claim 9, characterized in that: The applications include early identification and genotyping of the American strain of porcine reproductive and respiratory syndrome virus.

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