Primer group for detecting porcine reproductive and respiratory syndrome virus based on whole genome, library building method and application

Abstract

The invention provides a primer group for detecting a porcine reproductive and respiratory syndrome virus based on a whole genome, a library building method and application. The primer group comprises 16 primer pairs, and sequences of an upstream primer and a downstream primer of each primer pair are shown as SEQ ID NO.1-SEQ ID NO.32. The invention further provides a kit for detecting the porcine reproductive and respiratory syndrome virus based on the whole genome. The primer group disclosed by the invention has good specificity and sensitivity, and has a good application prospect in amplification of the whole genome of the porcine reproductive and respiratory syndrome virus and preparation of reagents or kits required by amplification. The library building method provided by the invention comprises the following steps: carrying out multiple PCR amplification by using the primer group provided by the invention; the library building method is suitable for a next-generation sequencing platform and a nanopore sequencing platform, the automation degree is high, 95% or above of porcine reproductive and respiratory syndrome virus sequences can be obtained even in a sample with the virus content as low as 1 copies / microliter, and the sequencing depth can reach 1000 X or above.

Description

Technical Field

[0001] The present invention relates to the field of high-throughput technology of viruses, and in particular to a primer set, a library construction method and an application for detecting porcine reproductive and respiratory syndrome virus based on the whole genome. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) Porcine Reproductive and Respiratory Syndrome, PRRS ) genotype 1 (PRRSV-1) and PRRSV genotype 2 (PRRSV-2) are now considered to be two different species, belonging to the Arteriviridae family along with 15 other virus species from primates, rodents, and equines. Arteriviridae ). The prototype strains of PRRSV-1 and PRRSV-2 (Lelystad and VR-2332) were discovered in Europe (PRRSV-1) and North America (PRRSV-2) around 1991, respectively, with a nucleotide sequence variation of about 44%. Today, these two species are widely distributed worldwide, with PRRSV-1 mainly distributed in Europe and PRRSV-2 mainly distributed in America and Asia. Genetic evolution analysis based on ORF5 showed that subtype 1 strains of PRRSV-1 can be divided into 12 different clades. Corresponding to the grouping of PRRSV-1 subtype 1, based on the genetic evolution analysis of the ORF5 gene, PRRSV-2 can be divided into 9 different lineages, of which 7 lineages are mainly prevalent in North America, and 2 lineages are only prevalent in East Asia. Similar to the evolutionary relationship between different subtypes of PRRSV-1, genetic evolution analysis shows that different lineages of PRRSV-2 may have diverged before PRRSV-2 was discovered in North America. Therefore, it is very likely that PRRSV-2 has begun to spread and evolve on a large scale before PRRS was identified as an emerging swine disease. PRRSV-2 in Asia may be mainly caused by the continuous mutation of the North American lineage after its introduction, leading to new outbreaks of the disease and increased virulence. Rapid differential diagnosis of PRRSV-2-infected samples and obtaining their full genome information can provide a basis for early detection, early control, and traceability of the virus, thereby minimizing the economic losses it brings to the pig industry.

[0003] At present, the main nucleic acid detection methods in virus detection are fluorescent quantitative PCR (qPCR), Sanger sequencing (first-generation sequencing) and high-throughput sequencing (second-generation sequencing and nanopore sequencing). qPCR can monitor the changes in fluorescence signals during PCR amplification in real time, realize quantitative analysis of target nucleic acids, quickly identify PRRSV infection, and is suitable for accurate determination of virus content. However, qPCR usually only detects specific porcine reproductive and respiratory syndrome virus gene fragments (such as ORF5 of envelope glycoprotein GP5), and cannot provide full genome information of porcine reproductive and respiratory syndrome virus. First-generation sequencing uses a sequencing-by-synthesis method to determine the DNA sequence by capturing special markers (usually fluorescent molecular markers) carried by newly added bases during DNA replication. It is suitable for small fragment sequence analysis with its high accuracy and long read length advantages. It can be combined with fluorescent quantitative PCR to achieve the qualitative and typing of PRRSV. With the continuous deepening of genomic research, the demand for sequencing throughput and speed is increasing. Fluorescent quantitative PCR and first-generation sequencing technologies are gradually unable to meet the needs of large-scale genome sequencing, so high-throughput sequencing technology came into being. High-throughput sequencing has rapidly become a mainstream technology for genomic research due to its high throughput, low cost and high speed. Therefore, providing a primer set suitable for whole genome sequencing of porcine reproductive and respiratory syndrome virus using high-throughput sequencing technology is of great significance for rapid diagnosis and typing of porcine reproductive and respiratory syndrome virus and whole genome research. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a primer set, a library construction method and application for whole genome detection of porcine reproductive and respiratory syndrome virus (PRRSV-2); the primer set of the present invention has good specificity and sensitivity, and has good application prospects in the amplification of the whole genome of porcine reproductive and respiratory syndrome virus and the preparation of reagents or kits required for amplification. The library construction method of the present invention is suitable for the second-generation sequencing platform and the nanopore sequencing platform, with a high degree of automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the porcine reproductive and respiratory syndrome virus sequences can be obtained, and the sequencing depth can reach more than 1000X; and when sequencing on the second-generation sequencing platform, the number of sequences per sample is only 5MB; when sequencing on the nanopore sequencing platform, the amount of data per sample is only 100MB.

[0005] The purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a primer set for detecting porcine reproductive and respiratory syndrome virus based on the whole genome, the primer set comprising 16 pairs of primers, the sequences of the upstream primers and the downstream primers of the 16 pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO. 32.

[0006] In some embodiments of the present invention, the 16 pairs of primers with sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 32 are divided into primer set 1 and primer set 2 at intervals, as shown in Table 4.

[0007] In a second aspect, the present invention provides a reagent for amplifying the whole genome of porcine reproductive and respiratory syndrome virus, wherein the reagent comprises the primer set described in the first aspect.

[0008] In some embodiments of the present invention, the reagents include two groups of reagents, one group of reagents includes the primer set 1 described in the first aspect, and the other group of reagents includes the primer set 2 described in the first aspect.

[0009] In a third aspect, the present invention provides a kit for amplifying the whole genome of porcine reproductive and respiratory syndrome virus, the kit comprising the primer set described in the first aspect or the reagent described in the second aspect.

[0010] In a fourth aspect, the present invention provides use of the primer set described in the first aspect, the reagent described in the second aspect, or the kit described in the third aspect in amplifying the whole genome of porcine reproductive and respiratory syndrome virus.

[0011] In a fifth aspect, the present invention provides a method for constructing a library of the whole genome of porcine reproductive and respiratory syndrome virus, the method comprising performing multiple PCR amplification using the primer set described in the first aspect, the reagent described in the second aspect, or the kit described in the third aspect.

[0012] In some embodiments of the invention, the method comprises the following steps: (1) Collect samples and extract nucleic acid from them; (2) reverse transcribing the nucleic acid extracted in step (1) to obtain cNDA; (3) performing multiple PCR amplification on the cNDA obtained by reverse transcription in step (2) using the primer set described in the first aspect, the reagent described in the second aspect, or the kit described in the third aspect; (4) Construction of sequencing library; (5) Sequencing on a machine; (6) Bioinformatics analysis

[0013] In some embodiments of the present invention, the sample of step (1) includes one or more of feces, intestinal tissue, blood, etc.

[0014] In some embodiments of the present invention, the method for extracting nucleic acid in step (1) comprises a magnetic bead method.

[0015] In some embodiments of the present invention, the length of the amplified product obtained by amplification in step (3) is between 1000 and 1400 bp, preferably 1200 bp.

[0016] In some embodiments of the present invention, the amplification procedure of step (3) is: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, denaturation at 60°C for 30 s, denaturation at 72°C for 90 s, for a total of 30 cycles; and extension at 72°C for 5 min.

[0017] In some embodiments of the present invention, the reaction reagents for amplification in step (3) include VAHTS PathogenDNA Multiplex PCR Mix, cDNA, ddH2O and the primer set described in the first aspect.

[0018] In some embodiments of the present invention, the amplification in step (3) is performed in two groups, i.e., the cNDA obtained by reverse transcription in step (2) is subjected to multiple PCR amplification using primer set 1 described in the first aspect and primer set 2 described in the first aspect, respectively, and then the amplification products are combined.

[0019] In some embodiments of the present invention, in one reaction reagent for amplification in step (3), each 25 μL of reaction reagent includes 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 5 μL of primer set 1 described in the first aspect; in another reaction reagent, each 25 μL of reaction reagent includes 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 5 μL of primer set 2 described in the first aspect.

[0020] In some embodiments of the present invention, the step (3) further comprises purifying the amplified product obtained by amplification.

[0021] In some embodiments of the present invention, the step (4) of constructing a sequencing library is performed on a next-generation sequencing platform; preferably, the step of constructing a sequencing library specifically comprises the following steps: fragment shearing, end repair & dA addition, adapter ligation, library amplification, single-stranded circularization, enzyme digestion, and DNB preparation; more preferably, the step of constructing a sequencing library further comprises purifying the product obtained in the adapter ligation step and / or purifying the product obtained in the enzyme digestion step.

[0022] In some embodiments of the present invention, the above purification includes purification using purification magnetic beads; preferably, the purification includes: 1) Mix the product to be purified with the magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed; 2) Add freshly prepared 80% ethanol to rinse the beads and carefully remove the supernatant; 3) Open the lid and dry the magnetic beads at room temperature for 5-10 min, add ddH2O to resuspend the dried magnetic beads, and recover the supernatant after the magnetic beads are completely adsorbed to obtain the purified product.

[0023] In some embodiments of the present invention, in step 1), the volume ratio of the product to be purified and the magnetic beads is (0.5-3):1.

[0024] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0025] In some embodiments of the present invention, the step (4) of constructing a sequencing library is performed on a nanopore sequencing platform; preferably, the step of constructing a sequencing library specifically comprises the following steps: end repair & dA addition, barcode ligation, sample mixing, and adapter ligation; more preferably, the step of constructing a sequencing library further comprises purifying the product obtained in the end repair & dA addition step and / or purifying the product obtained in the barcode ligation step and / or purifying the product obtained in the adapter ligation step.

[0026] In some embodiments of the present invention, the mixing of samples refers to mixing different samples (especially samples connected with barcodes obtained from samples from different sources through the above steps); preferably, the mixing of different samples is based on the mass of DNA in the samples, mixed in equal mass or in a specific ratio, and the total amount of DNA in the mixed samples is between 1 and 2 μg. Mixing the samples connected with barcodes and then testing the samples can reduce the number of sample tests in the subsequent steps of the library construction method, thereby shortening the time of library construction.

[0027] In some embodiments of the present invention, the purification method for purifying the product obtained in the end repair & dA addition step and / or the product obtained in the barcode ligation step comprises purification using purification magnetic beads; preferably, the purification method comprises the following steps: 1) Mix the product to be purified with the magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed; 2) Add freshly prepared 80% ethanol to rinse the beads and carefully remove the supernatant; 3) Keep the sample on the magnetic rack, open the lid and dry the magnetic beads at room temperature for 5-10 min, add ddH2O to resuspend the dried magnetic beads, and recover the supernatant after the magnetic beads are completely adsorbed to obtain the purified product.

[0028] In some embodiments of the present invention, in step 1), the volume ratio of the product to be purified and the magnetic beads is (0.5-3):1.

[0029] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0030] In some embodiments of the present invention, the purification method for purifying the product obtained in the linker connection step comprises purification using purification magnetic beads; preferably, the purification method comprises the following steps: Ⅰ) Mix the product to be purified with the magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed; Ⅱ) Add Wash Buffer to rinse the magnetic beads and discard the supernatant; III) Add the magnetic beads resuspended in elution buffer and incubate at room temperature for 5-10 min. Then transfer the reaction tube to a magnetic rack and let it stand. After the magnetic beads are completely adsorbed, aspirate and recover the supernatant to obtain the purified product.

[0031] In some embodiments of the present invention, in step I), the volume ratio of the product to be purified and the magnetic beads is (0.5-3):1.

[0032] In some embodiments of the present invention, step II) is repeated 1 to 3 times.

[0033] In some embodiments of the present invention, the Wash Buffer in step II) is a Wash Buffer used in conjunction with purification magnetic beads.

[0034] In some embodiments of the present invention, the elution buffer in step III) is an elution buffer used in conjunction with purification magnetic beads.

[0035] In some embodiments of the present invention, when purification magnetic beads are used for purification, the container containing 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.

[0036] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variation detection and evolution analysis.

[0037] The beneficial effects of the present invention are as follows: The present invention proposes a primer set for detecting porcine reproductive and respiratory syndrome virus based on the whole genome. The primer set is designed by a shingled primer design method. There is an overlapping region of 100 to 200 bp between adjacent primer pairs, which can ensure that the primer set can cover the entire genome. In addition, adjacent primer pairs are placed in different primer sets, and then two different primer sets are used to amplify and then merge the amplified products, which can effectively avoid non-specific binding of primers and improve amplification efficiency. The primer set of the present invention has good specificity and sensitivity, and has good application prospects in amplifying the whole genome of porcine reproductive and respiratory syndrome virus and preparing reagents or kits required for amplification.

[0038] The library construction method of the present invention is suitable for the second-generation sequencing platform and the nanopore sequencing platform, and has a high degree of automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the porcine reproductive and respiratory syndrome virus sequences can be obtained, and the sequencing depth can reach more than 1000X; and when sequencing on the second-generation sequencing platform, the number of sequences per sample only needs 5MB; when sequencing on the nanopore sequencing platform, the amount of data per sample only needs 100MB. Further, the present invention sets the amplicon size of each primer pair to 1000-1400bp, so that it can meet the requirements of the second-generation sequencing platform and the nanopore sequencing platform for the fragment size, thereby improving the compatibility of the library construction method with the sequencing platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the PRRSV whole genome targeted panel design of the present invention is shown.

[0040] Figure 2 The technical route of using the primer set of the present invention to perform whole genome sequencing of porcine reproductive and respiratory syndrome virus (PRRSV2) on a second-generation sequencing platform and a nanopore sequencing platform is demonstrated.

[0041] Figure 3 The whole genome coverage obtained by Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0042] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0043] Some of the experimental reagents used in the examples of the present invention are shown in Table 1: Table 1 Experimental reagents

[0044] Some of the experimental instruments and equipment used in the embodiments of the present invention are shown in Table 2: Table 2 Experimental instruments and equipment

[0045] The experimental samples used in the embodiments of the present invention are shown in Table 3: Table 3 Experimental samples

[0046] In Table 3: the sample corresponding to ZKJY-P3 originated from the pure culture of VR2332 strain, and the reference genome was EF536003.1; the sample corresponding to ZKJY-P4 originated from the pure culture of JXA1 strain, and the reference genome was EF112445.1; the sample corresponding to ZKJY-P5 originated from the pure culture of NADC30 strain, and the reference genome was JN654459.1; the sample corresponding to ZKJY-P6 originated from the pure culture of NADC34 strain, and the reference genome was MF326985.1; PK15 was a cell culture and was used as a negative control in the experiment.

[0047] The primer sets used in the embodiments of the present invention are shown in Table 4: Table 4 Primer set for PRRSV2 whole genome amplification

[0048] The design ideas of the primer sets in Table 4 above are as follows: referring to relevant domestic and foreign literature, summarizing all known PRRSV2 full genome sequences, removing duplicate sequences through multiple sequence alignment and sequence clustering, and selecting representative sequences as reference genomes for primer design, and then designing primers through shingled primer design (with 100-200 bp overlap regions between adjacent primer pairs) (primer design schematic diagram as shown in the figure). Figure 1 As shown in the figure), 15 pairs of primers covering the whole genome of PRRSV2 were finally obtained (where LEFT represents the upstream primer and RIGHT represents the downstream primer); in order to cover as much of the whole genome sequence as possible, two primers were added to the head and tail of the reference sequence after the design was completed; in order to cover as many variant strains as possible, there were different degrees of degenerate bases in the designed primers; adjacent primer pairs were placed in different primer groups to ensure specific amplification and whole genome coverage. The technical route for using the primer group of the present invention to sequence the whole genome of porcine reproductive and respiratory syndrome virus (PRRSV2) on the second-generation sequencing platform and the nanopore sequencing platform is as follows Figure 2 As shown, the specific process is as described in the following Examples 1 and 2.

[0049] Example 1 (Next Generation Sequencing) The experimental samples of this embodiment are: 4 kinds of nucleic acid samples ZKJY-P3, ZKJY-P4, ZKJY-P5, and ZKJY-P6 extracted from pure cultures of VR2332 strain, JXA1 strain, NADC30 strain, and NADC34 strain, respectively, and the nucleic acid concentration in the samples is 10000 copies / μL; ZKJY-P7, ZKJY-P8, ZKJY-P9, and ZKJY-P10 are all samples obtained by dilution of ZKJY-P6, and the nucleic acid concentrations in the samples are 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; a total of 8 samples, 3 replicates are set for each sample, and a total of 24 samples. The acquisition methods of ZKJY-P3, ZKJY-P4, ZKJY-P5, and ZKJY-P6 are as follows: 100 μL of pure culture of the strain was extracted by magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit of Thermo Fisher Scientific, and the nucleic acid was re-dissolved using 90 μL Elution Buffer to obtain 90 μL nucleic acid solution; then 5 μL of nucleic acid solution was taken for qPCR reaction using a PCR instrument (Applied Biosystems real-time fluorescence quantitative PCR instrument QuantStudio 1Plus, the same below), and the corresponding copy number was calculated using the obtained CT value; and the copy number was adjusted to 10,000 copies / μL. The library construction experiment process is as follows: 1. Reverse transcription: This process uses the UltraClean ds-cDNA Synthesis Module reverse transcription kit UNR201 of Nanjing Novozyme Biotechnology Co., Ltd. to synthesize cDNA from sample nucleic acid, as follows: 1-1. One-strand synthesis: Prepare the reagents according to Table 5 using the experimental samples (RNA) in Table 3, vortex to mix and centrifuge briefly, and place in a PCR instrument to react according to the program in Table 6.

[0050] Table 5 Reagents and dosages for single-strand synthesis

[0051] Table 6 PCR reaction program for one-strand synthesis

[0052] 1-2. Second-chain synthesis: Prepare the reagents according to Table 7, vortex to mix and centrifuge briefly, close the hot cover, place in the PCR instrument and perform the reaction according to the program in Table 8.

[0053] Table 7 Reagents and dosages for second-strand synthesis

[0054] Table 8 PCR reaction program for double strand synthesis

[0055] 2. PRRSV2 Whole Genome Amplification MiniAmp Plus Thermal Cycle PCR instrument and VAHTS Pathogen DNA Multiplex PCR Mix kit were used. The cDNA synthesized in step 1 was used as a template. The 16 pairs of primers in Table 4 (SEQ ID NO. 1 to SEQ ID NO. 32) were used to prepare amplification reagents according to Table 9. The mixture was shaken and centrifuged briefly. The mixture was placed in the PCR instrument and amplified by multiple PCR according to the procedure in Table 10. Among them, the reagents in Table 9 are two different sets of amplification reagents. One set of amplification reagents contains VAHTS Pathogen DNA Multiplex PCR Mix 12.5μL, cDNA 2.5μL, ddH2O 5μL, and primer set 1 5μL; the other set of amplification reagents contains VAHTS Pathogen DNA Multiplex PCR Mix 12.5μL, cDNA 2.5μL, ddH2O5μL, and primer set 2 5μL. Both sets of amplification reagents were used for multiple PCR amplification of the PRRSV2 whole gene according to the procedure in Table 10.

[0056] Table 9 Reagents and dosages for PRRSV2 whole genome amplification

[0057] Table 10 PCR reaction program for PRRSV2 whole genome amplification

[0058] After the PCR reaction program is completed, 25 μL of the amplified products of primer set 1 and primer set 2 are combined into one tube, 50 μL of VAHTS DNA Clean Beads are added to the tube, incubated at room temperature for 5 minutes, transferred to a magnetic stand and allowed to stand for 5 minutes, and the supernatant is discarded after the magnetic beads are completely adsorbed; then 200 μL of freshly prepared ethanol with a volume fraction of 80% is added and allowed to stand for 30 seconds, and the supernatant is discarded, and 200 μL of freshly prepared ethanol with a volume fraction of 80% is added to the reaction tube and allowed to stand for 30 seconds, and the supernatant is discarded; then the magnetic beads are opened and dried at room temperature for 8 minutes, 40 μL of ddH2O is added to resuspend the dried magnetic beads, the magnetic stand is removed and incubated at room temperature for 5 minutes, and then transferred to a magnetic stand and allowed to stand for 5 minutes, after the magnetic beads are completely adsorbed, the supernatant is transferred to a 0.2 mL centrifuge tube, and the concentration and purity of the nucleic acid are determined by Nanodrop2000 for the supernatant, and then temporarily stored in a -20°C refrigerator for use.

[0059] 3. Library Construction Use the MGIEasy Fast Enzyme Digestion Library Preparation Kit to take 150ng of the amplified product obtained in step 2 for library construction. According to the instructions of the MGIEasy Fast Enzyme Digestion Library Preparation Kit, prepare the reagents required for library construction in advance. Then, the amplified product is interrupted according to the required fragment size and end-repaired & dA is added, and then the double-stranded DNA library is obtained through adapter connection, product purification, and library amplification steps. Then, the double-stranded DNA library is quantified using the Equalbit 1×dsDNA HS Assay Kit, and the concentration of the double-stranded DNA library is adjusted to above 10ng / μL, and then single-stranded circularization, enzyme digestion, and product purification are performed to obtain the single-stranded DNA library. Then, the single-stranded DNA library is quantified using the Equalbit 1×ssDNA HS Assay Kit to confirm the library construction results (library concentration>1ng / μL is qualified). The specific steps for constructing a sequencing library are as follows: 3.1. Fragment disruption, end repair and dA addition The amplified product obtained in step 2 was mixed with other components according to the dosage in Table 11 (the dosage of dsDNA was 150 ng, and its volume was calculated based on the concentration measured in step 2), vortexed to mix and centrifuged briefly, and placed in a PCR instrument to react according to the program in Table 12.

[0060] Table 11 Reagents and dosages for fragment shearing, end repair and dA addition

[0061] Table 12 PCR reaction procedures for fragment disruption, end repair and dA addition

[0062] 3.2. Connector connection Prepare the reagents according to Table 13 and pipette 25 μL of the prepared reagents into the reaction tube after the reaction in step 3.1, shake to mix and centrifuge briefly, then place in a PCR instrument and react according to the program in Table 14.

[0063] Table 13 Reagents and dosages for connector connection

[0064] Table 14 PCR reaction program for adapter ligation

[0065] After the PCR reaction program is completed, 100 μL of VAHTS DNA Clean Beads is added to the reaction tube. After incubation at room temperature for 5 minutes, the reaction tube is transferred to a magnetic stand and allowed to stand for 5 minutes. After the magnetic beads are completely adsorbed, the supernatant is discarded; then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and allowed to stand for 30 seconds, and the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and allowed to stand for 30 seconds, and the supernatant is discarded; then the magnetic beads are opened and dried at room temperature for 8 minutes, 50 μL of ddH2O is added to resuspend the dried magnetic beads, the magnetic stand is removed and incubated at room temperature for 5 minutes, and the reaction tube is transferred to a magnetic stand and allowed to stand for 5 minutes. After the magnetic beads are completely adsorbed, the supernatant is aspirated and transferred to a new tube, and finally 48 μL of connection product is obtained.

[0066] 3.3. Library amplification Place the ligation product obtained in step 3.2 on a PCR instrument and react according to the program in Table 15.

[0067] Table 15 PCR reaction program for library amplification

[0068] 3.4. Single-strand circularization Prepare the reagents according to Table 16 and pipette 12 μL of the prepared reagents into the reaction tube after the reaction in step 3.3, shake to mix and centrifuge briefly, then place in a PCR instrument and react according to the program in Table 17.

[0069] Table 16 Reagents and dosages for single-chain cyclization

[0070] Table 17 PCR reaction program for single-stranded circularization

[0071] 3.5. Enzymatic digestion Prepare the reagents according to Table 18 and pipette 4 μL of the prepared reagents into the reaction tube after the reaction in step 3.4, shake to mix and centrifuge briefly, then place in a PCR instrument and react according to the program in Table 19.

[0072] Table 18 Reagents and dosages for enzyme digestion

[0073] Table 19 PCR reaction program for restriction digestion

[0074] After the PCR reaction program is completed, add 3 μL Exo Stop Buffer to the reaction tube, vortex and oscillate 3 times using a vortex oscillator VORTEX-5, each time for 3 seconds, and then centrifuge briefly to collect the reaction product to the bottom of the tube. Then, 100 μL of VAHTS DNA Clean Beads was added to the reaction tube. After incubation at room temperature for 5 min, the reaction tube was transferred to a magnetic stand and allowed to stand for 5 min. After the magnetic beads were completely adsorbed, the supernatant was discarded. Then, 200 μL of 80% ethanol was added to the reaction tube and allowed to stand for 30 s. After that, the supernatant was discarded. Then, 200 μL of 80% ethanol was added to the reaction tube and allowed to stand for 30 s. After that, the supernatant was discarded. Then, the magnetic beads were opened and dried at room temperature for 8 min. 20 μL of ddH2O was added to resuspend the dried magnetic beads. The magnetic stand was removed and incubated at room temperature for 5 min. The reaction tube was transferred to a magnetic stand and allowed to stand for 5 min. After the magnetic beads were completely adsorbed, the supernatant was aspirated and transferred to a new tube. Finally, 20 μL of single-stranded library was obtained.

[0075] 4.DNB preparation and sequencing According to the MGISEQ-G99RS High-throughput Sequencing Reagent Set (G99FCL PE150) manual, the required ssDNA library volume for DNB preparation was calculated. The amount of ssDNA library required for each DNB preparation system was 20 fmol. Then, the Equalbit 1×ssDNA HS Assay Kit was used to quantify DNBs, and the DNB concentration was confirmed to be ≥8ng / μL. The final total amount of DNB required for the machine was 40fmol. The sequencing was performed using the MGI DNBSEQ-G99RS gene sequencer. The sequence number of each sample was 5MB, and the sequencing time was 6 to 12h.

[0076] 4.1. Denaturation The single-stranded library obtained in step 3.5 was mixed with other components according to the dosage in Table 20, vortexed to mix and centrifuged briefly, and placed in a PCR instrument to react according to the program in Table 21.

[0077] Table 20 Denaturing reagents and dosage

[0078] Table 21 Denaturing PCR reaction program

[0079] 4.2.DNB preparation Prepare the reagents according to Table 22 and pipette 22 μL of the prepared reagents into the reaction tube after the reaction in step 4.1, shake and mix well, centrifuge briefly, and place in the PCR instrument to react according to the program in Table 23. When the temperature of the PCR instrument reaches 4°C, immediately add 10 μL of DNB stop buffer, mix well by pipetting slowly 6 times with a wide-mouth pipette tip, and place at 4°C for use before use.

[0080] Table 22 Reagents and dosages for DNB preparation

[0081] Table 23 PCR reaction program for DNB preparation

[0082] 4.3. Preparation of DNB loading reagents and sequencing Prepare the DNB loading system according to Table 24 and perform the sequencing experiment according to the instructions for use of the MGI DNBSEQ-G99RS gene sequencer.

[0083] Table 24 Reagents and dosages for preparation of DNB loading system

[0084] 5. Bioinformatics Analysis Fastpc software was used to perform quality control on the data, BWA software was used to align it to the reference genome, samtools software was used to convert the file format, the alignment results were statistically analyzed, and bamdst was used to calculate indicators such as coverage and sequencing depth.

[0085] The results obtained by the above-described method are shown in Table 25. From the results in Table 25, it can be seen that the primer set of the present invention has good specificity, good sensitivity, and a genome coverage greater than 95% (wherein the whole genome coverage is shown in FIG. Figure 3 The minimum detection limit at a depth of 1000X can reach 1 copies / μL.

[0086] Table 25 Second generation sequencing results

[0087] In Table 25: PK15 is a negative control. After targeted amplification, the measured DNA concentration is extremely low, indicating that the targeted amplification failed and the library cannot be constructed and sequenced.

[0088] Example 2 (Nanopore Sequencing) The experimental samples of this embodiment are: two nucleic acid samples ZKJY-P5 and ZKJY-P6 extracted from pure cultures of NADC30 strain and NADC34 strain, respectively, and the nucleic acid concentration in the samples is 10000 copies / μL; ZKJY-P7, ZKJY-P8, ZKJY-P9, and ZKJY-P10 are samples obtained by dilution of ZKJY-P6, and the nucleic acid concentrations in the samples are 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; a total of 6 samples, 3 replicates are set for each sample, and a total of 18 samples. The acquisition methods of ZKJY-P5 and ZKJY-P6 are as follows: 100 μL of pure culture of the strain is extracted by magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit of Thermo Fisher Scientific, and the nucleic acid is dissolved back using 90 μL Elution Buffer to obtain 90 μL nucleic acid solution; then 5 μL of nucleic acid solution is taken for qPCR reaction through a PCR instrument (Applied Biosystems real-time fluorescence quantitative PCR instrument QuantStudio 1Plus, the same below), and the corresponding copy number is calculated with the obtained CT value; and the copy number is adjusted to 10000 copies / μL. The library construction experiment process is as follows: First, the amplification product was obtained according to the method described in steps 1 to 2 of Example 1, and then the obtained amplification product was used to build the library according to the instructions of the CycloneSEQ library construction kit, including end repair & dA addition and product purification, barcode connection and product purification, sample mixing, adapter connection and product purification. The final library recovery rate was >10% and it was normal. The library quality was >1μg, which met the requirements of the machine. The CycloneSEQ sequencing kit and the MGI CycloneSEQ-WT02 sequencer were used for sequencing, and the sequencing time was 12h. The specific experimental process is as follows: 1.1. End repair & dA addition The components in Table 26 were mixed by vortexing and centrifuged briefly, and then placed in a PCR instrument to react according to the program in Table 27. The volume of the amplified product (double-stranded DNA) was calculated based on the concentration of double-stranded DNA in the amplified product.

[0089] Table 26 Reagents and dosages for end repair and dA addition

[0090] Table 27 PCR reaction program for end repair & addition of dA

[0091] After the PCR reaction program is completed, add 60 μL of VAHTS DNA Clean Beads to the reaction tube, incubate at room temperature for 5 minutes, transfer the reaction tube to the magnetic rack and let it stand for 5 minutes, and discard the supernatant after the magnetic beads are completely adsorbed; then add 200 μL of freshly prepared 80% ethanol by volume to the reaction tube and let it stand for 30 seconds, then discard the supernatant, and then add 200 μL of freshly prepared 80% ethanol by volume to the reaction tube and let it stand for 30 seconds, then discard the supernatant; then open the lid and dry the magnetic beads at room temperature for 8 minutes, add 27 μL ddH2O to resuspend the dried magnetic beads, remove the magnetic rack and incubate at room temperature for 5 minutes, then transfer the reaction tube to the magnetic rack and let it stand for 5 minutes, and after the magnetic beads are completely adsorbed, aspirate the supernatant and transfer it to a new tube, and finally obtain 25 μL of supernatant.

[0092] 1.2. Barcode connection Prepare the reagents according to Table 28 using the supernatant (dsDNA after end repair) obtained in step 1.1, vortex to mix and centrifuge briefly, then place in a PCR instrument and react according to the program in Table 29.

[0093] Table 28 Reagents and dosages for barcode connection

[0094] Table 29 PCR reaction program for barcode ligation

[0095] After the PCR reaction program is completed, 40 μL of VAHTS DNA Clean Beads is added to the reaction tube. After incubation at room temperature for 5 minutes, the reaction tube is transferred to a magnetic stand and allowed to stand for 5 minutes. After the magnetic beads are completely adsorbed, the supernatant is discarded; then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and allowed to stand for 30 seconds, and the supernatant is discarded. Then 200 μL of freshly prepared 80% ethanol is added to the reaction tube and allowed to stand for 30 seconds, and the supernatant is discarded; then the magnetic beads are opened and dried at room temperature for 8 minutes, 17 μL of ddH2O is added to resuspend the dried magnetic beads, the magnetic stand is removed and incubated at room temperature for 5 minutes, and the reaction tube is transferred to a magnetic stand and allowed to stand for 5 minutes. After the magnetic beads are completely adsorbed, the supernatant is aspirated and transferred to a new tube, and finally 15 μL of supernatant is obtained, and the DNA concentration is determined using the Equalbit 1×dsDNA HS Assay Kit.

[0096] 1.3. Mixed samples According to the total DNA input of 1.0 μg, the products obtained from the end repair, dA addition, product purification, barcode connection and product purification steps of the six samples ZKJY-P5, ZKJY-P6, ZKJY-P7, ZKJY-P8, ZKJY-P9 and ZKJY-P10 were mixed with equal mass according to the DNA mass (the volume required to be added to each sample during mixing is calculated according to the sample concentration determined in step 1.2), and the samples to be mixed were mixed into a 1.5 mL low adsorption tube, and after mixing, the total volume was filled with water to 125 μL.

[0097] 1.4. Connector connection Prepare the reagents according to Table 30 based on the product obtained by mixing the samples in step 1.3, vortex to mix and centrifuge briefly, then place in a PCR instrument and react according to the program in Table 31.

[0098] Table 30 Reagents and dosages for connector connection

[0099] Table 31 PCR reaction program for adapter ligation

[0100] After the PCR reaction program is completed, add 80μL of VAHTS DNA Clean Beads to the reaction tube. After incubation at room temperature for 5 minutes, transfer the reaction tube to the magnetic stand and let it stand for 5 minutes. After the magnetic beads are completely adsorbed, the supernatant is discarded; then add 300μL of Wash Buffer 1 used with magnetic beads (i.e., Wash Buffer 1 used with VAHTS DNA Clean Beads provided by Nanjing Novozymes Biotech Co., Ltd., the same below) to the reaction tube for elution, and the supernatant is discarded; then add 300μL of Wash Buffer 1 used with magnetic beads to the reaction tube for elution, and the supernatant is discarded; then add 42μL of elution buffer used with magnetic beads (i.e., Wash Buffer 1 used with VAHTS DNA Clean Beads provided by Nanjing Novozymes Biotech Co., Ltd.) Resuspend the magnetic beads in elution buffer used with Equalbit 1×dsDNA HS Assay Kit, remove the magnetic stand and incubate at room temperature for 7 minutes, then transfer the reaction tube to the magnetic stand and let it stand for 5 minutes. After the magnetic beads are completely adsorbed, transfer 40 μL of the supernatant to a new 1.5 mL centrifuge tube, and use the Equalbit 1×dsDNA HS Assay Kit to determine the DNA concentration in the supernatant.

[0101] 2. Sequencing Referring to the instructions of the CycloneSEQ sequencing kit, the product obtained in step 1.4 was sequenced using the CycloneSEQ sequencing kit and the MGI CycloneSEQ-WT02 gene sequencer.

[0102] 3. Bioinformatics Analysis Nanofilt software was used to perform quality control on the data, minimap software was used to align it to the reference genome, samtools software was used to convert the file format, the alignment results were statistically analyzed, bedtools software was used to generate bed files, and bamdst was used to calculate coverage, sequencing depth and other indicators.

[0103] The results obtained by the method described above are shown in Table 32. From the results in Table 32, it can be seen that the primer set of the present invention has good specificity, good sensitivity, and a genome coverage of greater than 95%, and the minimum detection limit of 1000X depth can reach 1 copies / μL.

[0104] Table 32 Nanopore sequencing results

[0105] In Table 32: PK15 is a negative control. After targeted amplification, the measured DNA concentration is extremely low, indicating that the targeted amplification failed and the library cannot be constructed and sequenced.

[0106] Example 3 (Clinical application of primer set) The experimental samples of this embodiment are: 2 nucleic acid samples ZKJY-P1 and ZKJY-P2 were extracted from two different tissues of the diseased material. The acquisition methods of ZKJY-P1 and ZKJY-P2 are: take 200μL of tissue grinds and use the automatic nucleic acid extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit of Thermo Fisher Scientific Biotechnology Co., Ltd. to extract nucleic acids by magnetic beads, use 90μL Elution Buffer to dissolve the nucleic acids, and obtain 90μL nucleic acid solution; then take 5μL of nucleic acid solution for qPCR reaction, and calculate the corresponding copy number with the obtained CT value. Then perform the library construction experiment according to the following steps: 1. Obtain the amplified product according to steps 1 to 2 of Example 1.

[0107] 2. The amplified product obtained in step 1 was subjected to steps 3 to 5 of Example 1 to obtain the next generation sequencing results, as shown in Table 33.

[0108] 3. The amplified product obtained in step 1 was subjected to steps 1 to 3 of Example 2 to obtain the nanopore sequencing results, as shown in Table 33.

[0109] It can be seen from the clinical sample test results in Table 33 that the primer set of the present invention has good specificity, good sensitivity, a genome coverage greater than 95%, and a sequencing depth greater than 1000X.

[0110] Table 33 Clinical sample sequencing results

[0111] In Table 33: the data volume of the second-generation sequencing is expressed in sequence numbers; the data volume of nanopore sequencing is expressed in the number of bases.

[0112] As can be seen from the above examples, the present invention provides a primer set, library construction method and application for detecting porcine reproductive and respiratory syndrome virus (PRRSV2) based on the whole genome. The primer set has good specificity and sensitivity, and the library construction method has adaptability, which can simplify manual operations, shorten time, and improve the detection efficiency of the whole genome sequence of PRRSV2. This shows that the present invention has successfully established a method for constructing a whole genome library of porcine reproductive and respiratory syndrome virus, and its executability has been verified through examples. It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A primer set for detecting porcine reproductive and respiratory syndrome virus based on the whole genome, characterized in that: The primer set includes 16 pairs of primers, and the sequences of the upstream primers and the downstream primers of the 16 pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO.

32.

2. A reagent for amplifying the whole genome of porcine reproductive and respiratory syndrome virus, characterized in that: The reagents include the primer set according to claim 1.

3. A kit for amplifying the whole genome of porcine reproductive and respiratory syndrome virus, characterized in that: The kit comprises the primer set according to claim 1 or the reagent according to claim 2.

4. Use of the primer set according to claim 1, the reagent according to claim 2, or the kit according to claim 3 in amplifying the whole genome of porcine reproductive and respiratory syndrome virus.

5. A method for constructing a library of the whole genome of porcine reproductive and respiratory syndrome virus, characterized in that: The method comprises performing multiplex PCR amplification using the primer set of claim 1 or the reagent of claim 2 or the kit of claim 3.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) Collect samples and extract nucleic acid from them; (2) reverse transcribing the nucleic acid extracted in step (1) to obtain cNDA; (3) performing multiplex PCR amplification on the cNDA obtained by reverse transcription in step (2) using the primer set of claim 1, the reagent of claim 2, or the kit of claim 3; (4) Construction of sequencing library; (5) Sequencing on a machine; (6) Bioinformatics analysis 7. The method according to claim 6, characterized in that The sample of step (1) includes one or more of feces, intestinal tissue, and blood; And / or, the method for extracting nucleic acid in step (1) comprises a magnetic bead method; And / or, the length of the amplified product obtained by amplification in step (3) is between 1000 and 1400 bp; And / or, the amplification procedure of step (3) is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, denaturation at 65°C for 30s, denaturation at 72°C for 90s, for a total of 30 cycles; extension at 72°C for 5min; And / or, the reaction reagents for amplification in step (3) include VAHTS Pathogen DNA Multiplex PCR Mix, gDNA, ddH2O and the primer set according to claim 1; And / or, the step (3) further comprises purifying the amplified product obtained by amplification; And / or, the step (4) of constructing a sequencing library may be performed on a second generation sequencing platform or a nanopore sequencing platform; And / or, the bioinformatics analysis in step (6) includes sequence alignment, variation detection and evolution analysis.

8. The method according to claim 7, characterized in that The step (4) of constructing a sequencing library is performed on a second-generation sequencing platform, and the construction of the sequencing library specifically includes the following steps: fragment shearing, end repair & dA addition, adapter ligation, library amplification, single-stranded circularization, enzyme digestion, and DNB preparation; Alternatively, the step (4) of constructing a sequencing library is performed on a nanopore sequencing platform, and the construction of the sequencing library specifically includes the following steps: end repair & dA addition, barcode ligation, sample mixing, and adapter ligation.

9. The method according to claim 8, characterized in that When the step (4) of constructing a sequencing library is performed on a next-generation sequencing platform, the step of constructing a sequencing library further includes purifying the product obtained in the adapter ligation step and / or purifying the product obtained in the enzyme digestion step; When the step (4) of constructing a sequencing library is performed on a nanopore sequencing platform, the step of constructing a sequencing library further includes purifying the product obtained from the end repair & dA addition step and / or purifying the product obtained from the barcode connection step and / or purifying the product obtained from the adapter connection step.

10. The method according to any one of claims 7 to 9, characterized in that: The purification includes purification using purification magnetic beads.

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