Primer group for detecting porcine epidemic diarrhea virus based on whole genome, library building method and application

Abstract

The invention provides a primer group for detecting a porcine epidemic diarrhea virus based on a whole genome, a library building method and application. The primer group comprises 27 pairs of primers, and sequences of upstream primers and downstream primers of the 27 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO.54. The invention further provides a kit for detecting the porcine epidemic diarrhea virus based on the whole genome. The primer group disclosed by the invention has good specificity, sensitivity and repeatability, and has a good application prospect in amplification of the whole genome of the porcine epidemic diarrhea 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 epidemic diarrhea 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 epidemic diarrhea virus based on the whole genome. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) Porcine Epidemic Diarrhea Virus, PEDV for short ) is a pathogen that causes porcine epidemic diarrhea and belongs to the Coronaviridae family ( Coronaviridae ), Coronavirus ( Coronavirus ). PEDV mainly infects pigs, especially piglets, causing severe diarrhea, vomiting and dehydration, usually accompanied by high mortality, causing significant economic losses to the pig industry. The genome of PEDV is a single-stranded positive-strand RNA with a length of approximately 28 kb; and the genome contains multiple open reading frames (ORFs) that can encode a variety of structural and non-structural proteins. The main structural proteins of PEDV include spike protein (S protein), membrane protein (M protein), nucleocapsid protein (N protein) and small membrane protein (E protein). Among them, the S protein plays a key role in the adsorption of the virus and invasion of host cells. The non-structural proteins of PEDV include the replicase complex (Rep) and accessory proteins (nsps), which play an important role in the replication and transcription of the virus.

[0003] At present, the nucleic acid detection methods in virus detection mainly include molecular diagnosis, Sanger sequencing (first generation sequencing) and high-throughput sequencing (second generation sequencing and nanopore sequencing). Molecular diagnosis includes reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (qPCR). RT-PCR is widely used in RNA virus detection (such as new coronavirus, influenza virus, etc.) by reverse transcribing RNA into cDNA and then performing PCR amplification. qPCR monitors the changes in fluorescence signals during PCR amplification in real time to achieve quantitative analysis of target nucleic acids, which is suitable for accurate determination of virus content. However, molecular diagnosis generally has the following disadvantages: 1. Limited detection range: RT-PCR and qPCR usually only detect specific gene fragments (such as N gene, S gene, etc.), and cannot provide full genome information of the virus; 2. False negative risk: If the gene of the virus in the primer region mutates, it may lead to false negative results; 3. Unable to provide virus mutation information: Molecular diagnostic methods cannot provide the complete genome sequence of the virus and cannot be used for virus mutation analysis and evolution research. Sanger sequencing, also known as first generation sequencing, is a DNA sequencing technology based on the dideoxynucleotide (ddNTP) chain termination method. By adding ddNTPs during DNA synthesis, the DNA chain is terminated at a specific position, thereby generating a series of DNA fragments of different lengths. These fragments are separated by electrophoresis, and the DNA sequence can be determined based on the fragment length and the type of terminating base. However, Sanger sequencing has the following disadvantages: 1. The cost is high, especially for large-scale genome sequencing, the cost of each sequencing reaction is high, and a large amount of reagents and manual operations are required; 2. The throughput is low, and it is difficult to meet the needs of large-scale genome sequencing. Each sequencing reaction can only read shorter DNA fragments; 3. It takes a long time, usually taking several days to complete the sequencing process, and is not suitable for rapid diagnosis and real-time analysis; 4. The degree of automation is low, requiring more manual operations, and it is difficult to achieve high-throughput automated sequencing.

[0004] At present, the high-throughput sequencing library construction methods for pathogen detection mainly rely on the library construction method based on the second-generation Illumina sequencing platform (two-round PCR) and the library construction method based on nanopore sequencing (long read length). However, these methods have some limitations in practical applications, such as the short read length of the second-generation sequencing, the difficulty of nanopore sequencing base quality reaching 99%, and the existing library construction methods are not universal between the two sequencing platforms, especially in the detection and research of porcine epidemic diarrhea virus (PEDV). Summary of the invention

[0005] 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 epidemic diarrhea virus (PEDV); the primer set of the present invention has good specificity, sensitivity and repeatability, and has good application prospects in the whole genome detection of porcine epidemic diarrhea virus and the preparation of reagents or kits required for detection. 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 porcine epidemic diarrhea 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 1MB; when sequencing on the nanopore sequencing platform, the amount of data per sample only needs 30MB.

[0006] 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 epidemic diarrhea virus based on the whole genome, the primer set comprising 27 pairs of primers, the sequences of the upstream primers and the downstream primers of the 27 pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO. 54.

[0007] In some embodiments of the present invention, the 27 pairs of primers with sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 54 are divided into primer set 1 and primer set 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 epidemic diarrhea virus, wherein the reagent comprises the primer set described in the first aspect.

[0009] 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.

[0010] In a third aspect, the present invention provides a kit for amplifying the whole genome of porcine epidemic diarrhea virus, the kit comprising the primer set described in the first aspect or the reagent described in the second aspect.

[0011] 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 whole genome amplification of porcine epidemic diarrhea virus.

[0012] In a fifth aspect, the present invention provides a method for constructing a whole genome library of porcine epidemic diarrhea 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.

[0013] 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

[0014] In some embodiments of the present invention, the sample of step (1) includes one or more of feces, intestinal tissue, anal swab, etc.

[0015] In some embodiments of the present invention, the method for extracting nucleic acid in step (1) comprises a magnetic bead method.

[0016] 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.

[0017] In some embodiments of the present invention, 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 2min, for a total of 35 cycles; and extension at 72°C for 2min.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments of the present invention, the step (3) further comprises purifying the amplified product obtained by amplification.

[0022] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variation detection and evolution analysis.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0027] In some embodiments of the present invention, the size of the fragments obtained after the fragmentation step is in the range of 150 to 350 bp.

[0028] 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.

[0029] 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.

[0030] 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) 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.

[0031] 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.

[0032] In some embodiments of the present invention, step 2) is repeated 1 to 3 times.

[0033] 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, remove the magnetic stand and incubate at room temperature for 5-10 min, then let it stand until the magnetic beads are completely adsorbed, then absorb and recover the supernatant to obtain the purified product.

[0034] 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.

[0035] In some embodiments of the present invention, step II) is repeated 1 to 3 times.

[0036] In some embodiments of the present invention, the Wash Buffer in step II) is a Wash Buffer used in conjunction with purification magnetic beads.

[0037] In some embodiments of the present invention, the elution buffer in step III) is an elution buffer used in conjunction with purification magnetic beads.

[0038] 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.

[0039] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variation detection and evolution analysis.

[0040] The beneficial effects of the present invention are as follows: The present invention proposes a primer set for detecting porcine epidemic diarrhea 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, sensitivity, and repeatability, and has good application prospects in amplifying the whole genome of porcine epidemic diarrhea virus and preparing reagents or kits required for amplification.

[0041] 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 porcine epidemic diarrhea 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 1MB; when sequencing on the nanopore sequencing platform, the amount of data per sample only needs 30MB. 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

[0042] Figure 1 The design principle of the primer set of the present invention (the correspondence between the primer pairs and the reference genome) is demonstrated.

[0043] Figure 2 The primer specificity and repeatability results obtained by Example 1 of the present invention are shown. In the figure, virus_panel1 contains 10 different viruses, including African swine fever virus, foot-and-mouth disease virus type A, foot-and-mouth disease virus type O, foot-and-mouth disease virus Asia type I, Japanese encephalitis virus, influenza A virus, porcine circovirus type 2, rotavirus, Geta virus and porcine gastroenteritis virus, but does not contain porcine epidemic diarrhea virus; virus_panel 2 contains 10 different viruses, including porcine epidemic diarrhea virus, porcine circovirus type 3, rotavirus type A, porcine parvovirus, porcine reproductive and respiratory syndrome virus type 1, porcine reproductive and respiratory syndrome virus type 2, pseudorabies virus, rabies virus, Seneca virus type A and porcine transmissible gastroenteritis virus.

[0044] Figure 3 The whole genome coverage obtained by Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0045] 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.

[0046] Some of the experimental reagents used in the examples of the present invention are shown in Table 1: Table 1 Experimental reagents

[0047] 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

[0048] The experimental samples used in the embodiments of the present invention are shown in Table 3: Table 3 Experimental samples

[0049] The primer sets used in the embodiments of the present invention are shown in Table 4: Table 4 Primer set for PEDV whole genome

[0050] The design ideas of the primer sets in Table 4 above are as follows: referring to relevant domestic and foreign literature, summarizing all known PEDV genome sequences, removing duplicate sequences through multiple sequence alignment and sequence clustering, and selecting representative sequences as reference genomes for primer design. Subsequently, primers were designed through a shingled primer design method (with an overlap region of 100 to 200 bp between adjacent primer pairs) (the corresponding relationship between primer pairs and reference genomes is shown in Figure 2). Figure 1 As shown in the figure), 27 primer pairs covering the entire PEDV genome were finally obtained (LEFT represents the upstream primer and RIGHT represents the downstream primer).

[0051] Example 1 (Next Generation Sequencing) The experimental samples of this embodiment are: nucleic acid samples extracted from pure virus culture of PEDV LW / L strain, the nucleic acid concentrations in the samples are: 1 copy / μL, 10 copies / μL, 100 copies / μL, 1000 copies / μL, and three replicates are set for each gradient concentration, for a total of 12 samples. The nucleic acid samples were obtained by taking 100 μL of pure virus culture of PEDV LW / L strain and using the nucleic acid extraction kit of Thermo Fisher Scientific Biotechnology Co., Ltd. through the magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit for nucleic acid extraction, and using 90 μL Elution Buffer to dissolve the nucleic acid to obtain 90 μL nucleic acid solution; then taking 5 μL of nucleic acid solution and passing it through a PCR instrument (Applied Biosystems' real-time fluorescence quantitative PCR instrument QuantStudio 1Plus, the same below) for qPCR reaction, and calculating the corresponding copy number with the obtained CT value; then diluting the original concentration of nucleic acid solution to nucleic acid concentrations of 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL, respectively, with three replicates for each concentration, and obtaining a total of 12 samples. 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 for the experimental samples (RNA) according to Table 5, vortex to mix and centrifuge briefly, and place in a PCR instrument to react according to the program in Table 6.

[0052] Table 5 Reagents and dosages for single-strand synthesis

[0053] Table 6 PCR reaction program for one-strand synthesis

[0054] 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.

[0055] Table 7 Reagents and dosages for second-strand synthesis

[0056] Table 8 PCR reaction program for double strand synthesis

[0057] 2. PEDV 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 primer set in Table 4 was used to prepare the amplification reagent according to the reagents and dosages in Table 9. The mixture was shaken and centrifuged briefly. The mixture was placed in the PCR instrument and the PEDV whole gene multiplex PCR amplification was performed according to the program 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, ddH2O 5μL, and primer set 2 5μL. Both sets of amplification reagents were used for PEDV whole gene multiplex PCR amplification according to the program in Table 10.

[0058] Table 9 Reagents and dosages for PEDV whole genome amplification

[0059] Table 10 PCR reaction program for PEDV whole genome amplification

[0060] 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, and 1 volume of VAHTS DNA Clean Beads is added to the 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, 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 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 transferred to a 1.5 mL centrifuge tube, and the supernatant is then used for Equalbit 1×ssDNA HS Assay The amplified products were quantified using the kit and the concentrations were determined to be above 50 ng / μL and stored in a -20°C refrigerator for later use.

[0061] 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 (double-stranded DNA) obtained in step 2 was mixed with other components according to the dosage in Table 11 (the dosage of double-stranded DNA 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.

[0062] Table 11 Reagents and dosages for fragment shearing, end repair and dA addition

[0063] Table 12 PCR reaction procedures for fragment disruption, end repair and dA addition

[0064] 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 and mix evenly, centrifuge briefly, and place in a PCR instrument to react according to the program in Table 14.

[0065] Table 13 Reagents and dosages for connector connection

[0066] Table 14 PCR reaction program for adapter ligation

[0067] After the PCR reaction program is completed, add 2 times the volume 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 50 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 1.5 mL new tube, and finally obtain 48 μL of connection product.

[0068] 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.

[0069] Table 15 PCR reaction program for library amplification

[0070] 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.

[0071] Table 16 Reagents and dosages for single-chain cyclization

[0072] Table 17 PCR reaction program for single-stranded circularization

[0073] 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 and mix evenly, centrifuge briefly, and place in a PCR instrument to react according to the program in Table 19.

[0074] Table 18 Reagents and dosages for enzyme digestion

[0075] Table 19 PCR reaction program for restriction digestion

[0076] 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, add 2 times the volume of VAHTS DNA Clean Beads to the reaction tube, incubate at room temperature for 5 minutes, transfer the reaction tube to a magnetic stand 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 20 μL ddH2O to resuspend the dried magnetic beads, remove the magnetic stand and incubate at room temperature for 5 minutes, then transfer the reaction tube to a magnetic stand and let it stand for 5 minutes, and after the magnetic beads are completely adsorbed, aspirate the supernatant and transfer it to a new 1.5 mL tube, and finally obtain 20 μL single-stranded library.

[0077] 4.DNB preparation and sequencing According to the instructions of the DNBSEQ-G99RS High-throughput Sequencing Reagent Set (G99FCL PE150), the required ssDNA library volume was calculated to prepare DNBs. The amount of ssDNA library required for each DNB preparation system was 20 fmol. Then, the Equalbit1×ssDNA HS Assay Kit was used to quantify DNBs and confirm that the DNB concentration was ≥8 ng / μL. The final total amount of DNB required for the machine was 40 fmol. The DNBSEQ-G99RS sequencer was used for sequencing. The sequence number of each sample was 1 MB, and the sequencing time was 6 to 12 hours.

[0078] 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.

[0079] Table 20 Denaturing reagents and dosage

[0080] Table 21 Denaturing PCR reaction program

[0081] 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.

[0082] Table 22 Reagents and dosages for DNB preparation

[0083] Table 23 PCR reaction program for DNB preparation

[0084] 4.3. DNB loading preparation 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.

[0085] Table 24 Reagents and dosages for preparation of DNB loading system

[0086] 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 and to count the alignment results, bedtools software was used to generate bed files, and bamdst was used to count indicators such as coverage and sequencing depth.

[0087] The results obtained by the above 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 of more than 95%. The minimum detection limit of 1000X depth can reach 1 copies / μL. In addition, the primer specificity and repeatability results obtained by the above method are shown in Table 25. Figure 2 The whole genome coverage is shown in Figure 3 shown.

[0088] Table 25 Second generation sequencing results

[0089] Example 2 (Nanopore Sequencing) The experimental samples of this embodiment are: nucleic acid samples extracted from pure virus culture of PEDV LW / L strain, the nucleic acid concentrations in the samples are: 1 copy / μL, 10 copies / μL, 100 copies / μL, 1000 copies / μL, and three replicates are set for each gradient concentration, for a total of 12 samples. The nucleic acid samples were obtained by taking 100 μL of pure virus culture of PEDV LW / L strain and using the nucleic acid extraction kit of Thermo Fisher Scientific Biotechnology Co., Ltd. to extract nucleic acid by magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit, and using 90 μL Elution Buffer to dissolve the nucleic acid to obtain 90 μL nucleic acid solution; then taking 5 μL of nucleic acid solution for qPCR reaction, and calculating the corresponding copy number with the obtained CT value; then diluting the original concentration of nucleic acid solution to the nucleic acid concentrations of 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL, respectively, with three replicates for each concentration, to obtain a total of 12 samples.

[0090] 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 gene sequencer were used for sequencing, and the sequencing time was 12h. The 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.

[0091] Table 26 Reagents and dosages for end repair and dA addition

[0092] Table 27 PCR reaction program for end repair & addition of dA

[0093] After the PCR reaction is completed, add 1 volume 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 1.5 mL tube, and finally obtain 25 μL of supernatant.

[0094] 1.2. Barcode connection Prepare the reagents according to Table 28 using the supernatant (end repair & dA addition product) obtained in step 1.1, vortex to mix, centrifuge briefly, and then place in a PCR instrument to react according to the program in Table 29.

[0095] Table 28 Reagents and dosages for barcode connection

[0096] Table 29 PCR reaction program for barcode ligation

[0097] 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 in the supernatant is determined using the Equalbit 1×dsDNA HS Assay Kit.

[0098] 1.3. Mixed samples According to the total DNA input of 1.2 μg, the products obtained from the end repair, dA addition, product purification, barcode connection and product purification steps of the four samples LW / L-1000, LW / L-100, LW / L-10 and LW / L-1 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). The samples to be mixed were mixed into a 1.5 mL low adsorption tube, and after mixing, water was added to a total volume of 125 μL.

[0099] 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.

[0100] Table 30 Reagents and dosages for connector connection

[0101] Table 31 PCR reaction program for adapter ligation

[0102] After the PCR reaction is completed, add 80μL (0.4X) 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., the same below) to the reaction tube for elution, and the supernatant is discarded. 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.

[0103] 1.5. 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.

[0104] 2. 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.

[0105] 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.

[0106] Table 32 Nanopore sequencing results

[0107] Example 3 (Clinical application of primer set) The experimental samples of this embodiment are: nucleic acid samples extracted from different types of samples in Table 3 (numbered 20230230, 20210284, 20230403, 20230498-5). The nucleic acid samples are obtained by taking 100 μL of sample and using the nucleic acid extraction kit of Thermo Fisher Scientific Biotechnology Co., Ltd. to extract nucleic acid by magnetic bead method using the automatic nucleic acid extractor KINGFISHERFLEX and MagMAX CORE Nucleic Acid Purification Kit, and using 90 μL ElutionBuffer to dissolve the nucleic acid to obtain 90 μL of nucleic acid solution; then take 5 μL of nucleic acid solution and perform qPCR reaction through a PCR instrument, and calculate the corresponding copy number with the obtained CT value. Then follow the steps below to perform the library construction experiment: 1. Obtain the amplified product according to steps 1 to 2 of Example 1.

[0108] 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.

[0109] 3. The amplified product obtained in step 1 was subjected to steps 1 to 2 of Example 2 to obtain the nanopore sequencing results, as shown in Table 33.

[0110] 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, and a genome coverage of more than 95%, and the minimum detection limit at a depth of 1000X can reach 1 copies / μL.

[0111] Table 33 Clinical sample sequencing results

[0112] Note: In Table 33: The data volume of the second-generation sequencing is expressed in the number of sequences; the data volume of nanopore sequencing is expressed in the number of bases.

[0113] As can be seen from the above examples, the present invention provides a primer set, a library construction method and an application for detecting porcine epidemic diarrhea virus based on the whole genome. The primer set has good specificity, sensitivity and repeatability, and the library construction method has adaptability, can simplify manual operation, shorten time, and improve the sequencing efficiency of the whole genome sequence of porcine epidemic diarrhea virus. This shows that the present invention has successfully established a whole genome library construction method for porcine epidemic diarrhea 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 epidemic diarrhea virus based on the whole genome, characterized in that: The primer set includes 27 pairs of primers, and the sequences of the upstream primers and downstream primers of the 27 pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO.

54.

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

3. A kit for amplifying the whole genome of porcine epidemic diarrhea 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 whole genome amplification of porcine epidemic diarrhea virus.

5. A method for constructing a whole genome library of porcine epidemic diarrhea 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 anal swab; 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 2min, for a total of 35 cycles; extension at 72°C for 2min; And / or, the reaction reagents for amplification in step (3) include VAHTS Pathogen DNA Multiplex PCR Mix, cDNA, 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.

Images