Primer set, library construction method and application based on whole genome detection of porcine reproductive and respiratory syndrome virus

Abstract

This invention provides a primer set, library construction method, and application for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV). The primer set comprises 16 primer pairs, and the sequences of the upstream and downstream primers for each primer pair are shown in SEQ ID NO. 1 to SEQ ID NO. 32. The primer set of this invention exhibits good specificity and sensitivity, and shows promising application prospects in the amplification of the entire PRSV genome and the preparation of reagents or kits required for amplification. The library construction method of this invention includes multiplex PCR amplification using the primer set described herein. This method is compatible with next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral concentrations as low as 1 copy / μL, it can obtain more than 95% of the PRSV sequence, with a sequencing depth exceeding 1000X.

Description

Technical Field

[0001] This invention relates to the field of high-throughput technology for viruses, and in particular to primer sets, library construction methods, and applications for whole-genome detection of porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) Porcine Reproductive and Respiratory Syndrome, Abbreviated as PRRS Genotype 1 (PRRSV-1) and genotype 2 (PRRSV-2) are now considered two distinct species, belonging to the Arteriviridae family along with 15 other viral species from primates, rodents, and equines. Arteriviridae The prototype strains of PRRSV-1 and PRRSV-2 (Lelystad and VR-2332) were discovered around 1991 in Europe (PRRSV-1) and North America (PRRSV-2), respectively, with a nucleotide sequence variability of approximately 44%. Today, both species are widely distributed globally, with PRRSV-1 primarily found in Europe and PRRSV-2 mainly in the Americas and Asia. Genetic evolution analysis based on ORF5 shows that PRRSV-1 subtype 1 can be divided into 12 distinct clades. Corresponding to the clades of PRRSV-1 subtype 1, genetic evolution analysis based on ORF5 genes divides PRRSV-2 into 9 distinct lineages, with 7 lineages predominantly circulating in North America and 2 lineages circulating only in East Asia. Similar to the evolutionary relationships among different subtypes of PRRSV-1, genetic evolutionary analysis suggests that different lineages of PRRSV-2 may have already diverged before its discovery in North America. Therefore, it is highly likely that PRRSV-2 had already begun widespread transmission and evolution before PRRS was recognized as a novel swine disease. The PRRSV-2 present in Asia may primarily have originated from the introduction of North American lineages and subsequent local mutations, leading to new outbreaks and increased virulence. Rapid differential diagnosis of PRRSV-2 infected samples and obtaining their whole-genome information can provide a basis for early virus detection, control, and source tracing, minimizing its economic losses to the swine industry.

[0003] Currently, nucleic acid detection methods for viruses mainly include quantitative real-time PCR (qPCR), Sanger sequencing (first-generation sequencing), and high-throughput sequencing (second-generation sequencing and nanopore sequencing). qPCR can monitor changes in fluorescence signals during PCR amplification in real time, enabling quantitative analysis of the target nucleic acid and rapid identification of PRRSV infection, suitable for accurate determination of viral load. However, qPCR typically only detects specific PRRSV gene fragments (such as ORF5 of the envelope glycoprotein GP5) and cannot provide whole-genome information of PRRSV. First-generation sequencing uses a sequencing-while-synthesizing method, determining the DNA sequence by capturing special markers (usually fluorescent molecular markers) carried by newly added bases during DNA replication. Its high accuracy and long read lengths make it suitable for small-fragment sequence analysis, and its combination with quantitative real-time PCR enables qualitative and genotyping of PRRSV. With the continuous deepening of genomics research, the demand for sequencing throughput and speed is increasing. Quantitative real-time PCR and first-generation sequencing technologies are gradually becoming insufficient to meet the needs of large-scale genome sequencing, leading to the emergence of high-throughput sequencing technology. High-throughput sequencing, with its advantages of high throughput, low cost, and high speed, has rapidly become the mainstream technology in genomics research. Therefore, providing a primer set suitable for whole-genome sequencing of porcine reproductive and respiratory syndrome virus (PRRSV) using high-throughput sequencing technology is of great significance for the rapid diagnosis, typing, and whole-genome research of PRSV. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide primer sets, library construction methods, and applications for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV-2). The primer sets of this invention possess excellent specificity and sensitivity, and show promising application prospects in the preparation of reagents or kits required for whole-genome amplification and preparation of PRRSV. The library construction method of this invention is compatible with both next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral concentrations as low as 1 copy / μL, over 95% of the PRRSV sequence can be obtained, with sequencing depths exceeding 1000X. Furthermore, when sequencing on a next-generation sequencing platform, only 5MB of sequence data is required per sample; when sequencing on a nanopore sequencing platform, only 100MB of data is required per sample.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides a primer set for detecting porcine reproductive and respiratory syndrome virus based on whole genome, the primer set comprising 16 pairs of primers, the sequences of the upstream and downstream primers of the 16 pairs of primers being shown in SEQ ID NO. 1 to SEQ ID NO. 32.

[0007] In some embodiments of the present invention, the 16 pairs of primers, as shown in SEQ ID NO. 1 to SEQ ID NO. 32, are divided into primer group 1 and primer group 2 at intervals, as shown in Table 4.

[0008] In a second aspect, the present invention provides a reagent for amplifying the whole genome of porcine reproductive and respiratory syndrome virus, the reagent comprising the primer set described in the first aspect.

[0009] In some embodiments of the present invention, the 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.

[0010] Thirdly, 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 reagents described in the second aspect.

[0011] Fourthly, the present invention provides the application 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 the whole genome amplification of porcine reproductive and respiratory syndrome virus.

[0012] Fifthly, the present invention provides a method for constructing a library of the whole genome of porcine reproductive and respiratory syndrome virus, the method comprising multiplex PCR amplification using the primer set described in the first aspect, the reagents described in the second aspect, or the kit described in the third aspect.

[0013] In some embodiments of the present invention, the method includes the following steps:

[0014] (1) Collect samples and extract nucleic acids from the samples;

[0015] (2) The nucleic acid extracted in step (1) is reverse transcribed to obtain cNDA;

[0016] (3) Perform multiplex 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;

[0017] (4) Construct sequencing libraries;

[0018] (5) Sequencing;

[0019] (6) Bioinformatics analysis.

[0020] In some embodiments of the present invention, the sample in step (1) includes one or more of feces, intestinal tissue, blood, etc.

[0021] In some embodiments of the present invention, the method for nucleic acid extraction in step (1) includes the magnetic bead method.

[0022] In some embodiments of the present invention, the length of the amplification product obtained in step (3) is between 1000 and 1400 bp, preferably 1200 bp.

[0023] In some embodiments of the present invention, the amplification procedure of step (3) 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 30 cycles; extension at 72°C for 5 minutes.

[0024] 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.

[0025] In some embodiments of the present invention, the amplification in step (3) is carried out in two groups, namely, multiplex PCR amplification of the cNDA obtained by reverse transcription in step (2) is performed using primer set 1 and primer set 2 described in the first aspect, respectively, and then the amplification products are combined.

[0026] In some embodiments of the present invention, each 25 μL of the reaction reagent in step (3) amplification includes 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 1.5 μL of the primer set described in the first aspect; the other 25 μL of the reaction reagent includes 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 2.5 μL of the primer set described in the first aspect.

[0027] In some embodiments of the present invention, step (3) further includes purifying the amplified product obtained by amplification.

[0028] In some embodiments of the present invention, step (4) 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 & dA addition, adapter ligation, library amplification, 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 enzyme digestion step.

[0029] In some embodiments of the present invention, the purification described above includes purification using purification magnetic beads; preferably, the purification includes:

[0030] 1) Mix the product to be purified with magnetic beads, and discard the supernatant after the magnetic beads have completely adsorbed the product;

[0031] 2) Add freshly prepared 80% ethanol to rinse the magnetic beads and carefully remove the supernatant;

[0032] 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.

[0033] 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.

[0034] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0035] In some embodiments of the present invention, step (4) of constructing the sequencing library is performed on a nanopore sequencing platform; preferably, the construction of the sequencing library specifically includes the following steps: end repair & dA addition, barcode ligation, sample mixing, and adapter ligation; more preferably, the step of constructing the sequencing library further includes purifying the product obtained from the end repair & dA addition step and / or purifying the product obtained from the barcode ligation step and / or purifying the product obtained from the adapter ligation step.

[0036] In some embodiments of the present invention, the sample mixing refers to mixing different samples (especially samples obtained from samples of different sources through the aforementioned steps and linked with barcodes); preferably, the mixing of different samples is based on the mass of DNA in the samples, mixing them in equal masses or in a specific proportion, and the total amount of DNA in the mixed samples is between 1 and 2 μg. Mixing the barcode-linked samples before sample detection can reduce the number of sample detection steps in the subsequent steps of the library construction method, thereby shortening the library construction time.

[0037] In some embodiments of the present invention, the purification method for purifying the product obtained from the end-repair & dA addition step and / or purifying the product obtained from the barcode linking step includes purification using purification magnetic beads; preferably, the purification method includes the following steps:

[0038] 1) Mix the product to be purified with magnetic beads, and discard the supernatant after the magnetic beads have completely adsorbed the product;

[0039] 2) Add freshly prepared 80% ethanol to rinse the magnetic beads and carefully remove the supernatant;

[0040] 3) Keep the sample on the magnetic rack, 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.

[0041] 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.

[0042] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0043] In some embodiments of the present invention, the purification method for purifying the product obtained in the connector connection step includes purification using purification magnetic beads; preferably, the purification method includes the following steps:

[0044] I) Mix the product to be purified with magnetic beads, and discard the supernatant after the magnetic beads have completely adsorbed the product;

[0045] Ⅱ) After adding Wash Buffer to rinse the magnetic beads, discard the supernatant;

[0046] 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 until the magnetic beads are completely adsorbed. Then aspirate and collect the supernatant to obtain the purified product.

[0047] In some embodiments of the present invention, the volume ratio of the product to be purified to the magnetic beads in step I) is (0.5-3):1.

[0048] In some embodiments of the present invention, step II) is repeated 1 to 3 times.

[0049] In some embodiments of the present invention, the Wash Buffer in step II) is a Wash Buffer used in conjunction with the purification magnetic beads.

[0050] In some embodiments of the present invention, the elution buffer in step III) is an elution buffer used in conjunction with the purification magnetic beads.

[0051] 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.

[0052] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variant detection and evolutionary analysis.

[0053] The beneficial effects of this invention are as follows:

[0054] This invention proposes a primer set for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV). This primer set was designed using a shingled primer design method, with 100-200 bp overlap between adjacent primer pairs, ensuring coverage of the entire genome. Furthermore, placing adjacent primer pairs in separate primer sets and then performing amplification using two different primer sets before merging the amplification products effectively avoids non-specific primer binding and improves amplification efficiency. The primer set of this invention exhibits good specificity and sensitivity, and shows promising application prospects in the amplification of the entire PRSV genome and the preparation of reagents or kits required for amplification.

[0055] The library preparation method of this invention is applicable to both next-generation sequencing (NGS) and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral loads as low as 1 copy / μL, it can obtain over 95% of the porcine reproductive and respiratory syndrome virus (PRRSV) sequence, with sequencing depths exceeding 1000X. Furthermore, when sequencing on a NGS platform, only 5MB of sequence is required per sample; when sequencing on a nanopore sequencing platform, only 100MB of data is required per sample. Further, this invention sets the amplicon size for each primer pair to 1000–1400 bp, ensuring it meets the fragment size requirements of both NGS and nanopore sequencing platforms, thereby improving the compatibility of the library preparation method with the sequencing platform. Attached Figure Description

[0056] Figure 1 A schematic diagram of the PRRSV whole-genome targeted panel design of this invention is shown.

[0057] Figure 2 This paper demonstrates a technical approach for whole-genome sequencing of porcine reproductive and respiratory syndrome virus (PRRSV2) using the primer set of this invention on both next-generation sequencing and nanopore sequencing platforms.

[0058] Figure 3 The whole genome coverage obtained by Example 1 of the present invention is shown. Detailed Implementation

[0059] 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.

[0060] Some of the experimental reagents used in the embodiments of this invention are shown in Table 1:

[0061] Table 1 Experimental Reagents

[0062]

[0063] Table 2 shows some of the experimental instruments and equipment used in the embodiments of this invention:

[0064] Table 2 Experimental Instruments and Equipment

[0065]

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

[0067] Table 3 Experimental Samples

[0068]

[0069] In Table 3: the sample corresponding to ZKJY-P3 was a pure culture of the VR2332 strain, with a reference genome of EF536003.1; the sample corresponding to ZKJY-P4 was a pure culture of the JXA1 strain, with a reference genome of EF112445.1; the sample corresponding to ZKJY-P5 was a pure culture of the NADC30 strain, with a reference genome of JN654459.1; the sample corresponding to ZKJY-P6 was a pure culture of the NADC34 strain, with a reference genome of MF326985.1; PK15 was a cell culture, used as a negative control in the experiment.

[0070] The primer sets used in the embodiments of this invention are shown in Table 4:

[0071] Table 4 Primer set used for PRRSV2 whole genome amplification

[0072]

[0073] The primer set design approach in Table 4 above is as follows: Referring to relevant domestic and international literature, all known PRRSV2 whole genome sequences were compiled. Repeated sequences were removed through multiple sequence alignment and sequence clustering. Representative sequences were selected as the reference genome for primer design. Subsequently, primers were designed using a shingled primer design method (adjacent primer pairs have a 100–200 bp overlap region). (A schematic diagram of the primer design is shown in Figure 4.) Figure 1 As shown in the figure, 15 primer pairs covering the entire PRRSV2 genome were finally obtained (where LEFT represents the upstream primer and RIGHT represents the downstream primer). To cover as much of the entire genome sequence as possible, two additional primers were added to the head and tail of the reference sequence after the design was completed. To cover as many variant strains as possible, the designed primers contained degenerate bases of varying degrees. Adjacent primer pairs were placed in different primer sets to ensure specific amplification and whole genome coverage. The technical route for whole genome sequencing of porcine reproductive and respiratory syndrome virus (PRRSV2) using the primer sets of this invention on next-generation sequencing platforms and nanopore sequencing platforms is as follows. Figure 2 As shown, the specific process is as described in Examples 1 and 2 below.

[0074] Example 1 (Next-Generation Sequencing)

[0075] The experimental samples in this embodiment were: four 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, with a nucleic acid concentration of 10,000 copies / μL; ZKJY-P7, ZKJY-P8, ZKJY-P9, and ZKJY-P10 were obtained by diluting ZKJY-P6, with nucleic acid concentrations of 1,000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; a total of 8 samples were used, with 3 replicates for each sample, for a total of 24 samples. ZKJY-P3, ZKJY-P4, ZKJY-P5, and ZKJY-P6 were obtained using the following method: 100 μL of pure culture of the virus strain was used to extract nucleic acids using the KINGFISHER FLEX automated nucleic acid extraction instrument and MagMAX CORE Nucleic Acid Purification Kit from Thermo Fisher Scientific via magnetic bead extraction. The nucleic acids were then reconstituted with 90 μL of Elution Buffer to obtain 90 μL of nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using a PCR instrument (Applied Biosystems' QuantStudio 1 Plus real-time fluorescence quantitative PCR instrument, hereinafter the same). The corresponding copy number was calculated based on the obtained CT value and adjusted to 10,000 copies / μL. The library construction experiment process is as follows:

[0076] 1. Reverse transcription: This process uses the UltraClean ds-cDNASynthesis Module Reverse Transcription Kit UNR201 from Nanjing Novizan Biotechnology Co., Ltd. to synthesize cDNA from the sample nucleic acids, as detailed below:

[0077] 1-1. One-chain synthesis:

[0078] Prepare the reagents according to Table 5 using the experimental samples (RNA) in Table 3, vortex to mix, and briefly centrifuge. Place the mixture in a PCR instrument and perform the reaction according to the procedure in Table 6.

[0079] Table 5. Reagents and dosages for one-chain synthesis

[0080]

[0081] Table 6 PCR reaction procedure for one-strand synthesis

[0082]

[0083] 1-2. Two-chain synthesis:

[0084] Prepare the reagents according to Table 7, vortex to mix, briefly centrifuge, close the hot cap, and place in a PCR instrument to perform the reaction according to the procedure in Table 8.

[0085] Table 7. Reagents and dosages for second-chain synthesis

[0086]

[0087] Table 8 PCR reaction procedure for second-strand synthesis

[0088]

[0089] 2. PRRSV2 whole genome amplification

[0090] Using a MiniAmp Plus Thermal Cycle PCR instrument and the VAHTS Pathogen DNA Multiplex PCR Mix kit, the cDNA synthesized in step 1 was used as a template. Amplification reagents were prepared according to Table 9 using the 16 primer pairs (SEQ ID NO. 1–SEQ ID NO. 32) listed in Table 4. The mixture was vortexed, briefly centrifuged, and then placed in the PCR instrument for PRRSV2 whole-gene multiplex PCR amplification according to the program in Table 10. Table 9 lists two different sets of amplification reagents: one set contained 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 1.5 μL of primer set 1; the other set contained 12.5 μL of VAHTS Pathogen DNA Multiplex PCR Mix, 2.5 μL of cDNA, 5 μL of ddH2O, and 2.5 μL of primer set 2. Both sets of reagents were used for PRRSV2 whole-gene multiplex PCR amplification according to the program in Table 10.

[0091] Table 9. Reagents and dosages for PRRSV2 whole genome amplification.

[0092]

[0093] Table 10 PCR reaction procedure for PRRSV2 whole genome amplification

[0094]

[0095] After the PCR reaction was completed, 25 μL of the amplification products from primer set 1 and primer set 2 were combined into one tube. 50 μL of VAHTS DNA Clean Beads were added to the tube, and the mixture was incubated at room temperature for 5 min. Then, it was transferred to a magnetic rack and allowed to stand for 5 min until the magnetic beads were completely adsorbed. The supernatant was then discarded. 200 μL of freshly prepared 80% ethanol was added and allowed to stand for 30 s. The supernatant was then discarded. Another 200 μL of freshly prepared 80% ethanol 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. 40 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. It 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 0.2 mL centrifuge tube. The concentration and purity of the nucleic acid in the supernatant were determined using Nanodrop 2000. The tube was then temporarily stored at -20°C for later use.

[0096] 3. Library Construction

[0097] Using the MGIEAsy Fast enzyme digestion library preparation kit, 150 ng of the amplification product obtained in step 2 was added for library construction. According to the MGIEAsy Fast enzyme digestion library preparation kit instructions, the reagents required for library construction were prepared in advance. Then, the amplification product was fragmented according to the required fragment size, and end repair and dA addition were performed. Following adapter ligation, product purification, and library amplification, a double-stranded DNA library was obtained. The double-stranded DNA library was then quantified using the Equalbit 1×dsDNA HS Assay Kit, adjusting the concentration to above 10 ng / μL. Single-stranded DNA was then circularized, digested, and purified to obtain a single-stranded DNA library. The single-stranded DNA library was then quantified using the Equalbit 1×ssDNA HS Assay Kit to confirm the library construction results (library concentration >1 ng / μL is considered acceptable). The specific steps for constructing the sequencing library are as follows:

[0098] 3.1. Fragment Breakdown and End-of-Fragment Repair & Adding dA

[0099] The amplification product obtained in step 2 is mixed with other components according to the amounts in Table 11 (the amount of dsDNA is 150 ng, and its volume is calculated based on the concentration obtained in step 2), vortexed and briefly centrifuged, and then placed in a PCR instrument to react according to the program in Table 12.

[0100] Table 11 Reagents and Doses for Fragment Segmentation and Terminal Repair & dA Addition

[0101]

[0102] Table 12 PCR reaction procedures for fragment fragmentation, end repair, and dA addition.

[0103]

[0104] 3.2. Connector Connection

[0105] Prepare the reagents according to Table 13 and add 25 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 reaction according to the procedure in Table 14.

[0106] Table 13. Reagents and dosages for connector connection.

[0107]

[0108] Table 14 PCR reaction procedures for adapter ligation

[0109]

[0110] After the PCR reaction program was completed, 100 μL of VAHTS 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 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 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. 50 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 48 μL of ligation product.

[0111] 3.3. Library Augmentation

[0112] The ligation product obtained in step 3.2 was placed on a PCR instrument and reacted according to the procedure in Table 15.

[0113] Table 15 PCR reaction procedure for library amplification

[0114]

[0115] 3.4. Single-chain cyclication

[0116] Prepare the reagents according to Table 16 and add 12 μL of the prepared reagents to the reaction tube after the reaction in step 3.3. Shake to mix and centrifuge briefly. Place the tube in a PCR instrument and perform the reaction according to the procedure in Table 17.

[0117] Table 16 Reagents and dosages for single-chain cyclization

[0118]

[0119] Table 17 PCR reaction procedure for single-stranded circularization

[0120]

[0121] 3.5. Enzymatic digestion

[0122] Prepare the reagents according to Table 18 and add 4 μL of the prepared reagents to the reaction tube after the reaction in step 3.4. Shake to mix and centrifuge briefly. Place the tube in a PCR instrument and perform the reaction according to the procedure in Table 19.

[0123] Table 18 Reagents and dosages for enzymatic digestion

[0124]

[0125] Table 19 PCR reaction procedure for enzyme digestion

[0126]

[0127] After the PCR reaction program is completed, add 3 μL of Exo Stop Buffer to the reaction tube, vortex 3 times for 3 seconds each time using a VORTEX-5 vortex mixer, and then briefly centrifuge 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 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 80% ethanol was added to the reaction tube and allowed to stand for 30 s. The supernatant was then discarded. Then, another 200 μL of 80% ethanol 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 single-stranded library.

[0128] 4. DNB preparation and sequencing

[0129] DNB was prepared according to the instructions of the MGISEQ-G99RS high-throughput sequencing kit (G99FCL PE150), calculating the required ssDNA library volume. Each DNB preparation system required 20 fmol of ssDNA library. DNB was then quantified using the Equalbit 1×ssDNA HS Assay Kit to confirm a concentration ≥8 ng / μL. The final DNB volume required for sequencing was 40 fmol. Sequencing was performed using the BGI Genomics DNBSEQ-G99RS gene sequencer, with each sample containing 5 MB of sequence data, and the sequencing time was 6–12 hours.

[0130] 4.1. Transgender

[0131] The single-stranded library obtained in step 3.5 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 reaction according to the procedure in Table 21.

[0132] Table 20 Denaturing reagents and dosages

[0133]

[0134] Table 21 PCR reaction procedure for denaturation

[0135]

[0136] 4.2. DNB Preparation

[0137] 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 reaction according to the program in Table 23. Once the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and gently pipette six times to mix. Store at 4°C before use.

[0138] Table 22 Reagents and dosages for DNB preparation

[0139]

[0140] Table 23 PCR reaction procedure for DNB preparation

[0141]

[0142] 4.3. DNB loading reagent preparation and sequencing

[0143] 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.

[0144] Table 24. Reagents and dosages used in the preparation of the DNB loading system.

[0145]

[0146] 5. Bioinformatics Analysis

[0147] Fastpc software was used for quality control of the sequencing data, BWA software was used for alignment to the reference genome, samtools software was used to convert the file format, the alignment results were statistically analyzed, and bamdst was used to statistically analyze indicators such as coverage and sequencing depth.

[0148] The results obtained using the methods described above are shown in Table 25. As can be seen from the results in Table 25, the primer set of this invention exhibits good specificity, high sensitivity, and genome coverage greater than 95% (the whole genome coverage diagram is shown below). Figure 3 As shown in the figure, the minimum detection limit at a depth of 1000X can reach 1 copy / μL.

[0149] Table 25. Second-generation sequencing results

[0150]

[0151] In Table 25: PK15 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.

[0152] Example 2 (Nanopore Sequencing)

[0153] The experimental samples in this embodiment are: two nucleic acid samples, ZKJY-P5 and ZKJY-P6, extracted from pure cultures of NADC30 and NADC34 strains, respectively, with a nucleic acid concentration of 10,000 copies / μL in each sample; ZKJY-P7, ZKJY-P8, ZKJY-P9, and ZKJY-P10 are all samples obtained by diluting ZKJY-P6, with nucleic acid concentrations of 1,000 copies / μL, 10 copies / μL, 10 copies / μL, and 1 copy / μL, respectively; a total of 6 samples, with 3 replicates for each sample, for a total of 18 samples. Both ZKJY-P5 and ZKJY-P6 were obtained as follows: 100 μL of pure culture of the strain was used to extract nucleic acids using the KINGFISHER FLEX automated nucleic acid extraction instrument and MagMAX CORE Nucleic Acid Purification Kit from Thermo Fisher Scientific via magnetic bead extraction. The nucleic acids were then reconstituted with 90 μL of Elution Buffer to obtain 90 μL of nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR reaction using a PCR instrument (Applied Biosystems QuantStudio 1 Plus real-time fluorescence quantitative PCR instrument, hereinafter the same). The corresponding copy number was calculated based on the obtained CT value and adjusted to 10,000 copies / μL. The library construction experiment process is as follows:

[0154] First, amplification products were obtained according to steps 1-2 of Example 1. Then, the obtained amplification products were used to construct a library according to the CycloneSEQ library construction kit instructions, including end repair & dA addition and product purification, barcode ligation and product purification, sample mixing, adapter ligation and product purification. A final library recovery rate >10% was considered normal, and the library mass >1 μg, meeting the requirements for sequencing. Sequencing was performed using the CycloneSEQ sequencing kit and the BGI Genomics CycloneSEQ-WT02 sequencer for 12 hours. The specific experimental procedure is as follows:

[0155] 1.1. End-point repair & dA addition

[0156] The components in Table 26 are shaken to mix and briefly centrifuged. The mixture is then placed in a PCR instrument and reacted according to the procedure in Table 27. The volume of the amplification product (double-stranded DNA) is calculated based on the concentration of double-stranded DNA in the amplification product.

[0157] Table 26 Reagents and Dosages for Terminal Repair & Adding dA

[0158]

[0159] Table 27 PCR reaction procedure for end repair & dA addition

[0160]

[0161] 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 min, then transfer the reaction tube to a magnetic rack and let it stand for 5 min until the magnetic beads are completely adsorbed, then discard the supernatant. Next, add 200 μL of freshly prepared 80% ethanol to the reaction tube, let it stand for 30 s, then discard the supernatant. Add another 200 μL of freshly prepared 80% ethanol to the reaction tube, let it stand for 30 s, then discard the supernatant. Then, open the cap and dry the magnetic beads at room temperature for 8 min, add 27 μL of ddH2O to resuspend the dried magnetic beads, remove the magnetic rack and incubate at room temperature for 5 min, then transfer the reaction tube to a magnetic rack and let it stand for 5 min until the magnetic beads are completely adsorbed, then aspirate the supernatant and transfer it to a new tube, finally obtaining 25 μL of supernatant.

[0162] 1.2. Barcode Connection

[0163] Using the supernatant (terminally repaired dsDNA) obtained in step 1.1, prepare the reagents according to Table 28, vortex to mix, and briefly centrifuge. Then place it in a PCR instrument and perform the reaction according to the program in Table 29.

[0164] Table 28 Reagents and Dosage for Barcode Links

[0165]

[0166] Table 29 shows the PCR reaction procedures linked by barcodes.

[0167]

[0168] After the PCR reaction program was completed, 40 μL of VAHTS 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 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 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 15 μL of supernatant. The DNA concentration was determined using the Equalbit 1×dsDNA HS Assay Kit.

[0169] 1.3. Mixed samples

[0170] With a total DNA input of 1.0 μg, the products obtained from six samples (ZKJY-P5, ZKJY-P6, ZKJY-P7, ZKJY-P8, ZKJY-P9, and ZKJY-P10) after end repair & dA addition and product purification, barcode ligation and product purification were mixed in equal mass using a DNA mass meter (the volume of each sample to be added during mixing was calculated based on the sample concentration obtained in step 1.2). The samples to be mixed were then mixed into 1.5 mL low-adsorption tubes, and water was added to bring the total volume to 125 μL.

[0171] 1.4. Connector Connection

[0172] Prepare reagents according to Table 30 using the product obtained from mixing in step 1.3, vortex to mix and briefly centrifuge, then place in a PCR instrument and react according to the procedure in Table 31.

[0173] Table 30. Reagents and dosages for connector connection.

[0174]

[0175] Table 31 PCR reaction procedure for adapter ligation

[0176]

[0177] After the PCR reaction program is complete, add 80 μL of VAHTS DNA Clean Beads to the reaction tube and incubate at room temperature for 5 min. Then, transfer the reaction tube to a magnetic rack and let it stand for 5 min until the magnetic beads are completely adsorbed, then discard the supernatant. Next, add 300 μL of Wash Buffer 1 (provided by Nanjing Novizan Biotechnology Co., Ltd.) to the reaction tube for elution, and discard the supernatant. Add another 300 μL of Wash Buffer 1 (provided by Nanjing Novizan Biotechnology Co., Ltd.) to the reaction tube for elution, and discard the supernatant. Finally, add 42 μL of elution buffer (provided by Nanjing Novizan Biotechnology Co., Ltd.) to the reaction tube for VAHTS DNA Clean Beads. Resuspend the magnetic beads in the elution buffer (used with the Beads), remove the magnetic rack and incubate at room temperature for 7 min. Then transfer the reaction tube to the magnetic rack and let it stand for 5 min. After the magnetic beads are completely adsorbed, aspirate 40 μL of supernatant and transfer it to a new 1.5 mL centrifuge tube. Use the Equalbit 1×dsDNA HS Assay Kit to determine the DNA concentration in the supernatant.

[0178] 2. Sequencing

[0179] Following the CycloneSEQ sequencing kit instructions, the products obtained in step 1.4 were sequenced using the CycloneSEQ sequencing kit and the BGI Genomics CycloneSEQ-WT02 gene sequencer.

[0180] 3. Bioinformatics Analysis

[0181] Nanofilt software was used for quality control of the sequencing data, minimap software was used for alignment to the reference genome, samtools software was used to convert the file format and analyze the alignment results, bedtools software was used to generate BED files, and bamdst was used to analyze metrics such as coverage and sequencing depth.

[0182] The results obtained by the methods described above are shown in Table 32. As can be seen from the results in Table 32, the primer set of the present invention has good specificity, good sensitivity, and genome coverage of more than 95%, and the lowest detection limit of 1000X can reach 1 copy / μL.

[0183] Table 32 Nanopore sequencing results

[0184]

[0185] In Table 32: PK15 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.

[0186] Example 3 (Clinical Application of Primer Kits)

[0187] The experimental samples in this embodiment were: two nucleic acid samples, ZKJY-P1 and ZKJY-P2, were extracted from two different tissues in the pathological sample. Both ZKJY-P1 and ZKJY-P2 were obtained as follows: 200 μL of the ground tissue was used to extract nucleic acids using the KINGFISHER FLEX automated nucleic acid extraction instrument and MagMAX CORE Nucleic Acid Purification Kit from Thermo Fisher Scientific via magnetic bead extraction. The nucleic acids were then reconstituted with 90 μL of Elution Buffer to obtain 90 μL of nucleic acid solution. Subsequently, 5 μL of the nucleic acid solution was used for qPCR, and the corresponding copy number was calculated based on the obtained CT value. Library construction experiments were then performed according to the following steps:

[0188] 1. Obtain the amplification product according to steps 1-2 of Example 1.

[0189] 2. The amplification products obtained in step 1 were processed according to steps 3 to 5 of Example 1 to obtain the next-generation sequencing results, as shown in Table 33.

[0190] 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 33.

[0191] As can be seen from the clinical sample test results in Table 33, the primer set of the present invention has good specificity, good sensitivity, genome coverage greater than 95%, and sequencing depth greater than 1000X.

[0192] Table 33 Sequencing results of clinical samples

[0193]

[0194] In Table 33: 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.

[0195] As demonstrated by the above embodiments, this invention provides a primer set, library construction method, and application for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV2). The primer set exhibits good specificity and sensitivity, and the library construction method is adaptable, simplifying manual operations, shortening time, and improving the detection efficiency of the PRRSV2 whole genome sequence. Therefore, this invention successfully establishes a method for whole-genome library construction of porcine reproductive and respiratory syndrome virus, and its feasibility has been verified through examples.

[0196] 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 detecting porcine reproductive and respiratory syndrome virus based on whole genome sequencing, characterized in that, The primer set includes 16 pairs of primers, and the sequences of the upstream and 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 reagent comprises the primer set as described in claim 1.

3. The reagent according to claim 2, characterized in that, The 16 pairs of primers, as shown in SEQ ID NO. 1 to SEQ ID NO. 32, are sequentially divided into primer set 1 and primer set 2. The reagents include two sets of reagents, one set of reagents including primer set 1 and the other set of reagents including primer set 2.

4. A kit for whole-genome amplification of porcine reproductive and respiratory syndrome virus, characterized in that, The kit includes the primer set as described in claim 1 or the reagent as described in claim 2 or 3.

5. The application of the primer set of claim 1, the reagent of claim 2 or 3, or the kit of claim 4 in the whole genome amplification of porcine reproductive and respiratory syndrome virus, wherein the application is not for diagnostic purposes.

6. A method for constructing a whole genome library of porcine reproductive and respiratory syndrome virus (PRRSV) for non-diagnostic purposes, characterized in that, The method includes performing multiplex PCR amplification using the primer set of claim 1, the reagent of claim 2 or 3, or the kit of claim 4.

7. The method according to claim 6, characterized in that, The 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 cNDA; (3) Perform multiplex PCR amplification on the cNDA obtained by reverse transcription in step (2) using the primer set described in claim 1, the reagent described in claim 2 or 3, or the kit described in claim 4; (4) Construct sequencing libraries; (5) Sequencing; (6) Bioinformatics analysis.

8. The method according to claim 7, characterized in that, The sample in step (1) includes one or more of the following: feces, intestinal tissue, and blood. And / or, the method for nucleic acid extraction in step (1) includes the magnetic bead method; And / or, the length of the amplification product obtained in step (3) is between 1000 and 1400 bp; And / or, the amplification program in step (3) is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 65℃ denaturation for 30s, 72℃ denaturation for 90s, for a total of 30 cycles; 72℃ extension for 5min; And / or, the amplification reaction reagents in step (3) include VAHTS Pathogen DNA Multiplex PCR Mix, gDNA, ddH2O, and the primer set as described in claim 1; And / or, step (3) further includes purifying the amplification product obtained from the amplification; And / or, step (4) 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 sequence alignment, variant detection and evolutionary analysis.

9. The method according to claim 7 or 8, characterized in that, The 16 pairs of primers, as shown in SEQ ID NO .1 to SEQ ID NO .32, are sequentially divided into primer set 1 and primer set 2. The amplification in step (3) is carried out in two groups. The nucleic acid extracted in step (1) is amplified by multiplex PCR using primer set 1 and primer set 2 respectively, and then the amplification products are combined.

10. The method according to claim 8, characterized in that, The amplification product obtained in step (3) is 1200 bp in length.

11. The method according to claim 8, characterized in that, The construction of the sequencing library in step (4) is carried out on the second-generation sequencing platform. The construction of the sequencing library specifically includes the following steps: fragment fragmentation, end repair & dA addition, adapter ligation, library amplification, single-strand circularization, enzyme digestion, and DNB preparation. Alternatively, step (4) 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 dA addition, barcode ligation, sample mixing, and adapter ligation.

12. The method according to claim 11, characterized in that, When step (4) of constructing the sequencing library is performed on a second-generation sequencing platform, the step of constructing the sequencing library also includes purifying the product obtained from the adapter ligation step and / or purifying the product obtained from the enzyme digestion step. When step (4) of constructing the sequencing library is performed on a nanopore sequencing platform, the step of constructing the sequencing library also includes purifying the product obtained from the end repair and dA addition steps and / or purifying the product obtained from the barcode ligation step and / or purifying the product obtained from the adapter ligation step.

13. The method according to any one of claims 8, 10, 11, and 12, characterized in that, The purification process includes purification using purification magnetic beads.

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