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

Abstract

The present invention provides a primer set, a library construction method and an application for detecting porcine epidemic diarrhea virus based on whole genome. 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 as SEQ ID NO. 1 to SEQ ID NO. 54. The primer set of the present invention has good specificity, sensitivity and repeatability, and has good application prospects in the amplification of the whole genome of porcine epidemic diarrhea virus and the preparation of reagents or reagent kits required for amplification. The library construction method of the present invention includes performing multiplex PCR amplification using the primer set described in the present invention; the library construction method of the present invention is adapted to second-generation sequencing platforms and nanopore sequencing platforms, with high automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the porcine epidemic diarrhea virus sequences can be obtained, and the sequencing depth can reach more than 1000X.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput of viruses, and particularly relates to a primer set, a library construction method and an application for detecting porcine epidemic diarrhea virus based on whole genome. Background Art

[0002] Porcine epidemic diarrhea virus ( Porcine Epidemic Diarrhea Virus, abbreviated as PEDV ) is a pathogen that causes porcine epidemic diarrhea and belongs to the family Coronaviridae ( Coronaviridae ) and the genus Coronavirus ( Coronavirus ). PEDV mainly infects pigs, especially piglets, causing severe diarrhea, vomiting and dehydration, usually accompanied by a high mortality rate, which causes significant economic losses to the pig industry. The genome of PEDV is a single-stranded positive-sense RNA with a length of about 28 kb; and the genome contains multiple open reading frames (ORFs), which can encode a variety of structural proteins 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 and invasion of host cells by the virus. The non-structural proteins of PEDV include replicase complex (Rep) and accessory proteins (nsps), and these proteins play important roles in the replication and transcription processes 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 quantitative fluorescence PCR (qPCR). RT-PCR reverses transcribes RNA into cDNA and then performs PCR amplification, which is widely used in the detection of RNA viruses (such as the novel coronavirus, influenza virus, etc.). qPCR monitors the change of fluorescence signal during the PCR amplification process in real time to achieve quantitative analysis of the target nucleic acid, and is suitable for the 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 the whole genome information of the virus; 2. False negative risk: If the gene of the virus mutates in the primer region, it may lead to false negative results; 3. Unable to provide virus variation information: Molecular diagnosis methods cannot provide the complete genome sequence of the virus and cannot be used for virus variation analysis and evolutionary research. Sanger sequencing, also known as first-generation sequencing, is a DNA sequencing technology based on the chain termination method of dideoxynucleotides (ddNTP). By adding ddNTP during DNA synthesis, the DNA chain terminates at specific positions, thus generating a series of DNA fragments with different lengths. These fragments are separated by electrophoresis, and the DNA sequence can be determined according to the fragment length and the type of terminating base. However, Sanger sequencing has the following disadvantages: 1. High cost, 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. Low throughput, difficult to meet the needs of large-scale genome sequencing, and each sequencing reaction can only read short DNA fragments; 3. Time-consuming, usually taking several days to complete the sequencing process, not suitable for rapid diagnosis and real-time analysis; 4. Low degree of automation, requiring more manual operations and difficult to achieve high-throughput automated sequencing.

[0004] At present, the high-throughput sequencing library construction methods for pathogenic microorganism detection mainly rely on the library construction methods based on the second-generation Illumina sequencing platform (two rounds of PCR) and the library construction methods based on nanopore sequencing (long read lengths). However, these methods have some limitations in practical applications. For example, the reads of second-generation sequencing are short, the base quality of nanopore sequencing is difficult to reach 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] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a primer set, a library construction method and an application for detecting Porcine Epidemic Diarrhea Virus (PEDV) based on whole genome; 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 adapted to the second-generation sequencing platform and the nanopore sequencing platform, with high automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the Porcine Epidemic Diarrhea Virus sequences can be obtained, and the sequencing depth can reach more than 1000X; when sequencing on the second-generation sequencing platform, the sequence number of each sample only needs 1MB; when sequencing on the nanopore sequencing platform, the data volume of each sample only needs 30MB.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In the first aspect, the present invention provides a primer set for detecting Porcine Epidemic Diarrhea Virus based on whole genome. 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.

[0008] In some embodiments of the present invention, the 27 pairs of primers with sequences 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.

[0009] In the second aspect, the present invention provides a reagent for amplifying the whole genome of Porcine Epidemic Diarrhea Virus, and the reagent includes the primer set described in the first aspect.

[0010] In some embodiments of the present invention, the reagent includes two groups of reagents, one group of reagents includes primer set 1 described in the first aspect, and the other group of reagents includes primer set 2 described in the first aspect.

[0011] In the third aspect, the present invention provides a kit for amplifying the whole genome of Porcine Epidemic Diarrhea Virus, and the kit includes the primer set described in the first aspect or the reagent described in the second aspect.

[0012] In the fourth aspect, 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 amplification of the whole genome of Porcine Epidemic Diarrhea Virus.

[0013] In the fifth aspect, the present invention provides a method for constructing a whole genome library of Porcine Epidemic Diarrhea Virus, and the method includes performing multiplex 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.

[0014] In some embodiments of the present invention, the method comprises the following steps:

[0015] (1) Collect a sample and perform nucleic acid extraction on the sample;

[0016] (2) Reverse transcribe the nucleic acid obtained in step (1) to obtain cDNA;

[0017] (3) Perform multiplex PCR amplification on the cDNA 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;

[0018] (4) Construct a sequencing library;

[0019] (5) Perform sequencing on a machine;

[0020] (6) Perform bioinformatics analysis.

[0021] In some embodiments of the present invention, the sample in step (1) includes one or more of feces, intestinal tissue, anal swab, etc.

[0022] In some embodiments of the present invention, the method for nucleic acid extraction in step (1) includes the magnetic bead method.

[0023] In some embodiments of the present invention, the length of the amplification product obtained by amplification in step (3) is between 1000 and 1400 bp, preferably 1200 bp.

[0024] In some embodiments of the present invention, the amplification program in step (3) is: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s, 65 °C for 30 s, 72 °C for 2 min, for a total of 35 cycles; extension at 72 °C for 2 min.

[0025] 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.

[0026] In some embodiments of the present invention, the amplification in step (3) is carried out in two groups, that is, multiplex PCR amplification is performed on the cDNA obtained by reverse transcription in step (2) using the primer set 1 described in the first aspect and the primer set 2 described in the first aspect respectively, and then the amplification products are combined.

[0027] In some embodiments of the present invention, in each 25 μL of a reaction reagent in the amplification of step (3), the 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, in each 25 μL of the reaction reagent, it 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.

[0028] In some embodiments of the present invention, step (3) further includes purifying the amplified product obtained by amplification.

[0029] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variant detection, and evolutionary analysis.

[0030] In some embodiments of the present invention, the construction of the sequencing library in step (4) is carried out on a next-generation sequencing platform; preferably, the construction of the sequencing library specifically includes the following steps: fragment fragmentation, end repair & addition of dA, adapter ligation, library amplification, single-strand circularization, enzymatic digestion, DNB preparation; more preferably, the steps of constructing the sequencing library further include purifying the product obtained in the adapter ligation step and / or purifying the product obtained in the enzymatic digestion step.

[0031] In some embodiments of the present invention, the above purification includes purification using purification magnetic beads; preferably, the purification includes:

[0032] 1) Mix the product to be purified with magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed.

[0033] 2) Add freshly prepared ethanol with a volume fraction of 80% to rinse the magnetic beads, and carefully remove the supernatant.

[0034] 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, that is, the purified product is obtained.

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

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

[0037] In some embodiments of the present invention, the fragment size obtained after the fragment fragmentation step is in the range of 150 - 350 bp.

[0038] In some embodiments of the present invention, step (4) of constructing the sequencing library is carried out on a nanopore sequencing platform; preferably, the construction of the sequencing library specifically includes the following steps: end repair & dA addition, barcode ligation, pooling, and adapter ligation; more preferably, the steps of constructing the sequencing library further include 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.

[0039] In some embodiments of the present invention, the pooling refers to mixing different samples (especially the barcoded samples obtained from samples with different sources through the foregoing steps); preferably, the different samples are mixed in equal mass or in a specific ratio based on the mass of DNA in the samples, and the total amount of DNA in the mixed samples is between 1 and 2 μg. Pooling the barcoded samples and then detecting the samples can reduce the number of sample detections in the subsequent steps of the library construction method, thereby shortening the time for library construction.

[0040] 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 ligation step includes using purification magnetic beads for purification; preferably, the purification method includes the following steps:

[0041] 1) Mix the product to be purified with the magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed.

[0042] 2) Add freshly prepared ethanol with a volume fraction of 80% to rinse the magnetic beads, and carefully remove the supernatant.

[0043] 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, that is, the purified product is obtained.

[0044] 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 - 3):1.

[0045] In some embodiments of the present invention, step 2) is repeated 1 - 3 times.

[0046] In some embodiments of the present invention, the purification method for purifying the product obtained from the adapter ligation step includes using purification magnetic beads for purification; preferably, the purification method includes the following steps:

[0047] Ⅰ) Mix the product to be purified with the magnetic beads, and discard the supernatant after the magnetic beads are completely adsorbed.

[0048] II) After adding Wash Buffer to rinse the magnetic beads, aspirate and discard the supernatant.

[0049] III) Add magnetic beads resuspended in elution buffer, remove from the magnetic stand and incubate at room temperature for 5 - 10 min, then let stand. After the magnetic beads are completely adsorbed, aspirate and recover the supernatant to obtain the purified product.

[0050] 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.

[0051] In some embodiments of the present invention, step II) is repeated 1 - 3 times.

[0052] In some embodiments of the present invention, the Wash Buffer in step II) is the Wash Buffer used in combination with the purified magnetic beads.

[0053] In some embodiments of the present invention, the elution buffer in step III) is the elution buffer used in combination with the purified magnetic beads.

[0054] In some embodiments of the present invention, when using purified magnetic beads for purification, during the purification process, the container containing the product to be purified and the magnetic beads is placed on the magnetic stand so that the magnetic beads can be adsorbed by magnetic force during the purification process.

[0055] In some embodiments of the present invention, the bioinformatics analysis in step (6) includes sequence alignment, variant detection, and evolutionary analysis.

[0056] The beneficial effects of the present invention are as follows:

[0057] The present invention provides a primer set for detecting porcine epidemic diarrhea virus based on whole - genome. This primer set is designed by the overlapping primer design method, and there is an overlapping region of 100 - 200 bp between adjacent primer pairs, which can ensure that the primer set can cover the entire genome. In addition, by placing adjacent primer pairs in different primer sets respectively, and then using two different primer sets for amplification and combining the amplification products, non - specific binding of primers can be effectively avoided and the amplification efficiency can be improved. The primer set of the present invention has good specificity, sensitivity, and repeatability, and has good application prospects in the amplification of the whole genome of porcine epidemic diarrhea virus, the preparation of reagents or kits required for amplification.

[0058] The library construction method of the present invention is applicable to second-generation sequencing platforms and nanopore sequencing platforms, with a high degree of automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the porcine epidemic diarrhea virus sequences can be obtained, and the sequencing depth can reach more than 1000X. Moreover, when sequencing on a second-generation sequencing platform, the sequence number of each sample only needs to be 1MB; when sequencing on a nanopore sequencing platform, the data volume of each sample only needs to be 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 fragment size, thereby improving the adaptability of the library construction method to the sequencing platform. Description of the Drawings

[0059] Figure 1 Shows the design principle of the primer set of the present invention (the corresponding relationship between primer pairs and the reference genome).

[0060] Figure 2 Shows the primer specificity and repeatability results obtained from Example 1 of the present invention. 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 I type, Japanese encephalitis virus, influenza A virus, porcine circovirus type 2, rotavirus, Getah virus, and porcine gastroenteritis virus, and 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.

[0061] Figure 3 Shows the whole-genome coverage obtained from Example 1 of the present invention. Detailed Embodiments

[0062] The following further illustrates the technology of the present invention through examples. These examples are the explanations and illustrations of the present invention and do not limit the scope of the present invention in any form.

[0063] Some of the experimental reagents used in the examples of the present invention are shown in Table 1:

[0064] Table 1 Experimental Reagents

[0065]

[0066] Some of the experimental instrument and equipment used in the examples of the present invention are shown in Table 2:

[0067] Table 2 Experimental Instrument and Equipment

[0068]

[0069] The experimental samples used in the embodiments of the present invention are shown in Table 3 as follows:

[0070] Table 3 Experimental Samples

[0071]

[0072] The primer sets used in the embodiments of the present invention are shown in Table 4 as follows:

[0073] Table 4 Primer Sets for the Complete Genome of PEDV

[0074]

[0075] The design idea of the primer sets in Table 4 above: Refer to relevant domestic and foreign literature, summarize all known complete genome sequences of PEDV, remove repetitive sequences through multiple sequence alignment and sequence clustering, and select representative sequences as the reference genome for primer design. Subsequently, primer design is carried out by the overlapping primer design method (with an overlapping region of 100 - 200 bp between adjacent primer pairs) (the corresponding relationship between primer pairs and the reference genome is as Figure 1 shown), and finally 27 pairs of primer pairs covering the complete genome of PEDV are obtained (where LEFT represents the upstream primer and RIGHT represents the downstream primer).

[0076] Example 1 (Next-generation Sequencing)

[0077] The experimental samples in this example are: nucleic acid samples extracted from the pure virus culture of the PEDV LW / L strain. The nucleic acid concentrations in the samples are: 1 copies / μL, 10 copies / μL, 100 copies / μL, 1000 copies / μL. Three replicate samples are set for each gradient concentration, totaling 12 samples. The acquisition method of the nucleic acid samples is as follows: Take 100 μL of the pure virus culture of the PEDV LW / L strain, and use the nucleic acid extraction kit of Thermo Fisher Scientific and the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit to extract nucleic acid by the magnetic bead method. Use 90 μL of Elution Buffer to redissolve the nucleic acid to obtain 90 μL of nucleic acid solution; then take 5 μL of the nucleic acid solution and perform qPCR reaction through a PCR instrument (the real-time fluorescence quantitative PCR instrument QuantStudio 1Plus of Applied Biosystems, the same below) to calculate the corresponding copy number based on the obtained CT value; then dilute the original concentration nucleic acid solution to nucleic acid concentrations of 1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copies / μL respectively, with three replicate samples for each concentration, obtaining a total of 12 samples. The process of library construction is as follows:

[0078] 1. Reverse transcription: This process uses the UltraClean ds-cDNA Synthesis Module reverse transcription kit UNR201 of Nanjing Novoprotein Scientific Co., Ltd. to synthesize cDNA from the sample nucleic acid, specifically as follows:

[0079] 1-1. First-strand synthesis:

[0080] Prepare the reagents for the experimental samples (RNA) according to Table 5, shake and mix well, briefly centrifuge, and place them in the PCR instrument to react according to the program in Table 6.

[0081] Table 5 Reagents and dosages for first-strand synthesis

[0082]

[0083] Table 6 PCR reaction program for first-strand synthesis

[0084]

[0085] 1-2. Second-strand synthesis:

[0086] Prepare the reagents according to Table 7, shake and mix well, briefly centrifuge, close the hot lid, and place them in the PCR instrument to react according to the program in Table 8.

[0087] Table 7 Reagents and Dosages for Double-Strand Synthesis

[0088]

[0089] Table 8 PCR Reaction Program for Double-Strand Synthesis

[0090]

[0091] 2. Amplification of the Full Genome of PEDV

[0092] Using the MiniAmp Plus Thermal Cycle PCR instrument and the VAHTS Pathogen DNA Multiplex PCR Mix kit, with the cDNA synthesized in step 1 as the template, prepare the amplification reagents according to the primers in Table 4 and the reagents and dosages in Table 9. Mix well by shaking and centrifuge briefly, then place in the PCR instrument and perform multiplex PCR amplification of the full genome of PEDV 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 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; the other set of amplification reagents contains 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. Both sets of amplification reagents are subjected to multiplex PCR amplification of the full genome of PEDV according to the program in Table 10.

[0093] Table 9 Reagents and Dosages for Amplification of the Full Genome of PEDV

[0094]

[0095] Table 10 PCR Reaction Program for Amplification of the Full Genome of PEDV

[0096]

[0097] After the PCR reaction program is completed, 25 μL of the amplification products of primer set 1 and primer set 2 are combined into one tube. Add 1 volume of VAHTS DNA Clean Beads to the tube, incubate at room temperature for 5 min, then transfer the reaction tube to a magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, discard the supernatant; then add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s, and then discard the supernatant. Add another 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s, and then discard the supernatant; then dry the magnetic beads with the lid open at room temperature for 8 min, add 40 μL of ddH2O to resuspend the dried magnetic beads, remove the tube from the magnetic stand and incubate at room temperature for 5 min, then transfer the reaction tube to the magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, aspirate the supernatant and transfer it to a 1.5 mL centrifuge tube. Then quantify the supernatant using the Equalbit 1×ssDNA HS Assay Kit to ensure that the concentration of the amplification products is above 50 ng / μL, and store it temporarily in a -20°C refrigerator for later use.

[0098] 3. Library construction

[0099] Use the MGIEasy Fast Enzyme Digestion Library Preparation Reagent Kit and add 150 ng of the amplification product obtained in step 2 to construct the library. According to the instructions of the MGIEasy Fast Enzyme Digestion Library Preparation Reagent Kit, prepare the reagents required for library construction in advance. Then fragment the amplification product according to the required fragment size and perform end repair & dA addition, followed by adapter ligation, product purification, and library amplification steps to obtain a double-stranded DNA library. Then use the Equalbit 1×dsDNA HS Assay Kit to quantify the double-stranded DNA library, adjust the concentration of the double-stranded DNA library to be above 10 ng / μL, and then perform single-stranded circularization, enzymatic digestion, and product purification to obtain a single-stranded DNA library. Then use the Equalbit 1×ssDNA HS Assay Kit to quantify the single-stranded DNA library to confirm the library construction results (a library concentration > 1 ng / μL is qualified). The specific steps for constructing the sequencing library are as follows:

[0100] 3.1. Fragmentation and end repair & dA addition

[0101] Mix the amplification product (double-stranded DNA) obtained in step 2 with other components according to the amounts in Table 11 (the amount of double-stranded DNA is 150 ng, and its volume is calculated based on the concentration determined in step 2), shake well and centrifuge briefly, and place it in a PCR instrument to react according to the program in Table 12.

[0102] Table 11 Reagents and amounts for fragmentation and end repair & dA addition

[0103]

[0104] PCR reaction program for fragment interruption, end repair & dA addition in Table 12

[0105]

[0106] 3.2. Adapter ligation

[0107] Prepare the reagents according to Table 13, pipette 25 μL of the prepared reagent into the reaction tube after the reaction in step 3.1, mix well by shaking and centrifuge briefly, then place it in the PCR instrument and carry out the reaction according to the program in Table 14.

[0108] Reagents and dosages for adapter ligation in Table 13

[0109]

[0110] PCR reaction program for adapter ligation in Table 14

[0111]

[0112] After the PCR reaction program is completed, add 2 volumes of VAHTS DNA Clean Beads to the reaction tube, incubate at room temperature for 5 min, then transfer the reaction tube to the magnetic rack and let it stand for 5 min. After the magnetic beads are completely adsorbed, discard the supernatant; then add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s and then discard the supernatant. Add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube again, let it stand for 30 s and then discard the supernatant; then dry the magnetic beads with the lid open at room temperature for 8 min, add 50 μL of ddH2O to resuspend the dried magnetic beads, remove the reaction tube from the magnetic rack and incubate at room temperature for 5 min, then transfer the reaction tube to the magnetic rack and let it stand for 5 min. After the magnetic beads are completely adsorbed, pipette the supernatant and transfer it to a new 1.5 mL tube to finally obtain 48 μL of the ligation product.

[0113] 3.3. Library amplification

[0114] Place the ligation product obtained in step 3.2 on the PCR instrument and carry out the reaction according to the program in Table 15.

[0115] PCR reaction program for library amplification in Table 15

[0116]

[0117] 3.4. Single-strand circularization

[0118] Prepare the reagents according to Table 16, pipette 12 μL of the prepared reagent into the reaction tube after the reaction in step 3.3, mix well by shaking and centrifuge briefly, then place it in the PCR instrument and carry out the reaction according to the program in Table 17.

[0119] Table 16 Reagents and Dosages for Single-Strand Cyclization

[0120]

[0121] Table 17 PCR Reaction Program for Single-Strand Cyclization

[0122]

[0123] 3.5. Restriction Digestion

[0124] 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. Mix well by shaking and centrifuge briefly, then place it in the PCR instrument and carry out the reaction according to the program in Table 19.

[0125] Table 18 Reagents and Dosages for Restriction Digestion

[0126]

[0127] Table 19 PCR Reaction Program for Restriction Digestion

[0128]

[0129] After the PCR reaction program is completed, add 3 μL of Exo Stop Buffer to the reaction tube, vortex it 3 times with a VORTEX-5 vortex oscillator for 3 s each time, and then centrifuge instantaneously to collect the reaction products to the bottom of the tube. Subsequently, add 2 volumes 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. After the magnetic beads are completely adsorbed, discard the supernatant; then add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s and then discard the supernatant. Add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube again, let it stand for 30 s and then discard the supernatant; then dry the magnetic beads at room temperature with the lid open for 8 min, add 20 μL of ddH2O to resuspend the dried magnetic beads, remove it from the magnetic rack and incubate at room temperature for 5 min, then transfer the reaction tube to the magnetic rack and let it stand for 5 min. After the magnetic beads are completely adsorbed, pipette the supernatant into a new 1.5 mL tube to finally obtain 20 μL of single-strand library.

[0130] 4. DNB Preparation and Sequencing on the Machine

[0131] Calculate the required volume of ssDNA library to prepare DNB according to the reagent instructions of the DNBSEQ-G99RS high-throughput sequencing reagent kit (G99FCL PE150). The amount of ssDNA library required for each DNB preparation system is 20 fmol. Then use the Equalbit1×ssDNA HS Assay Kit to quantify the DNB, confirm that the DNB concentration is ≥8 ng / μL, and the final requirement for the total amount of DNB for loading is 40 fmol. Use the DNBSEQ-G99RS sequencer for sequencing, with the number of sequences per sample being 1 MB and the sequencing duration being 6-12 hours.

[0132] 4.1. Denaturation

[0133] Mix the single-stranded library obtained in step 3.5 with other components according to the amounts in Table 20, shake well and centrifuge briefly, and place it in a PCR instrument to react according to the program in Table 21.

[0134] Table 20 Reagents and amounts for denaturation

[0135]

[0136] Table 21 PCR reaction program for denaturation

[0137]

[0138] 4.2. DNB preparation

[0139] 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 well and centrifuge briefly, and place it in a PCR instrument to react according to the program in Table 23. Immediately add 10 μL of DNB termination buffer when the temperature of the PCR instrument reaches 4°C, and gently pipette and mix 6 times with a wide-mouth pipette tip. It can be placed at 4°C for standby before use.

[0140] Table 22 Reagents and amounts for DNB preparation

[0141]

[0142] Table 23 PCR reaction program for DNB preparation

[0143]

[0144] 4.3. DNB loading preparation and sequencing on the machine

[0145] Prepare the DNB loading system according to Table 24 and perform the sequencing experiment according to the instructions for the MGI DNBSEQ-G99RS gene sequencer.

[0146] Table 24 Reagents and amounts for DNB loading system preparation

[0147]

[0148] 5. Bioinformatics analysis

[0149] The off-machine data was quality-controlled using the Fastpc software, aligned to the reference genome using the BWA software, the file format was converted using the samtools software, the alignment results were statistically analyzed, a bed file was generated using the bedtools software, and indicators such as coverage and sequencing depth were statistically analyzed using bamdst.

[0150] The results obtained by the above-described method are shown in Table 25. From the results in Table 25, it can be seen that the primer set of the present invention has good specificity, good sensitivity, a genome coverage of greater than 95%, and the lowest detection limit at a depth of 1000X can reach 1 copies / μL. In addition, the primer specificity and repeatability results obtained by the above-described method are as Figure 2 shown, and the obtained whole-genome coverage is as Figure 3 shown.

[0151] Table 25 Second-generation sequencing results

[0152]

[0153] Example 2 (Nanopore sequencing)

[0154] The experimental samples in this example were: nucleic acid samples extracted from the pure virus culture of the PEDV LW / L strain, with nucleic acid concentrations in the samples being: 1 copies / μL, 10 copies / μL, 100 copies / μL, 1000 copies / μL. Three replicates were set for each gradient concentration, for a total of 12 samples. The method for obtaining the nucleic acid samples was as follows: 100 μL of the pure virus culture of the PEDV LW / L strain was taken, and the nucleic acid was extracted using the nucleic acid extraction kit of Thermo Fisher Scientific Co., Ltd. by the magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and the MagMAX CORE Nucleic Acid Purification Kit. The nucleic acid was redissolved with 90 μL of Elution Buffer to obtain 90 μL of nucleic acid solution; then 5 μL of the nucleic acid solution was taken for qPCR reaction, and the corresponding copy number was calculated based on the obtained CT value; then the nucleic acid solution with the original concentration was diluted to nucleic acid concentrations of 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copies / μL respectively, with three replicates for each concentration, resulting in a total of 12 samples.

[0155] First, obtain the amplification product according to the method described in steps 1 to 2 of Example 1. Then, use the obtained amplification product to construct a library according to the instructions of the CycloneSEQ Library Construction Kit, including end repair & dA addition and product purification, barcode ligation and product purification, pooling, adapter ligation and product purification. Finally, a normal library recovery rate is >10%, and the library quality is >1 μg, meeting the requirements for loading onto the machine. Use the CycloneSEQ Sequencing Kit and the MGI CycloneSEQ-WT02 Gene Sequencer for sequencing, and the sequencing duration is 12 h. The specific experimental process is as follows:

[0156] 1.1. End repair & dA addition

[0157] Vortex and briefly centrifuge the components in Table 26, and place them in a PCR instrument to perform the reaction according to the program in Table 27. Among them, the volume of the amplification product (double-stranded DNA) is calculated according to the concentration of double-stranded DNA in the amplification product.

[0158] Table 26 Reagents and dosages for end repair & dA addition

[0159]

[0160] Table 27 PCR reaction program for end repair & dA addition

[0161]

[0162] After the PCR reaction is completed, add 1 volume of VAHTS DNA Clean Beads to the reaction tube. Incubate at room temperature for 5 min, then transfer the reaction tube to a magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, discard the supernatant; then add 200 μL of freshly prepared 80% ethanol by volume to the reaction tube, let it stand for 30 s, and then discard the supernatant. Add 200 μL of freshly prepared 80% ethanol by volume to the reaction tube again, let it stand for 30 s, and then discard the supernatant; then dry the magnetic beads with the lid open at room temperature for 8 min, add 27 μL of ddH2O to resuspend the dried magnetic beads, remove the reaction tube from the magnetic stand and incubate at room temperature for 5 min, then transfer the reaction tube to the magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, aspirate the supernatant and transfer it to a new 1.5 mL tube. Finally, obtain 25 μL of supernatant.

[0163] 1.2. Barcode ligation

[0164] Prepare the reagents according to Table 28 with the supernatant obtained in step 1.1 (end repair & dA addition product), vortex and briefly centrifuge, and then place them in a PCR instrument to perform the reaction according to the program in Table 29.

[0165] Table 28 Reagents and dosages for barcode ligation

[0166]

[0167] Table 29 PCR reaction procedure for barcode ligation

[0168]

[0169] After the PCR reaction procedure is completed, add 40 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 stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant. Subsequently, add 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s, then aspirate and discard the supernatant. Add another 200 μL of freshly prepared ethanol with a volume fraction of 80% to the reaction tube, let it stand for 30 s, and aspirate and discard the supernatant. Then, open the lid and dry the magnetic beads at room temperature for 8 min. Add 17 μL of ddH2O to resuspend the dried magnetic beads, remove the tube from the magnetic stand, incubate at room temperature for 5 min, then transfer the reaction tube back to the magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, aspirate the supernatant and transfer it to a new tube. Finally, obtain 15 μL of supernatant and measure the DNA concentration in the supernatant using the Equalbit 1×dsDNA HS Assay Kit.

[0170] 1.3. Sample mixing

[0171] According to a total DNA input of 1.2 μg, perform equal-mass sample mixing (by DNA mass) on the products obtained from the four samples of LW / L-1000, LW / L-100, LW / L-10, and LW / L-1 after end repair & dA addition and product purification, barcode ligation, and product purification steps. (The volume required for each sample during mixing is calculated based on the sample concentration determined in step 1.2.) Mix the samples to be mixed into a 1.5 mL low-binding tube, and after mixing, add water to make the total volume up to 125 μL.

[0172] 1.4. Adapter ligation

[0173] Prepare the reagents according to Table 30 using the product obtained from step 1.3. Vortex and mix well, then centrifuge briefly. Then place it in a PCR instrument and perform the reaction according to the program in Table 31.

[0174] Table 30 Reagents and dosages for adapter ligation

[0175]

[0176] Table 31 PCR reaction procedure for adapter ligation

[0177]

[0178] After the PCR reaction is completed, add 80 μL (0.4X) of VAHTS DNA Clean Beads to the reaction tube. Incubate at room temperature for 5 min, then transfer the reaction tube to a magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, discard the supernatant; Subsequently, add 300 μL of Wash Buffer1 used in conjunction with the magnetic beads (i.e., Wash Buffer1 provided by Nanjing Novoprotein Science & Technology Co., Ltd. and used in conjunction with VAHTS DNA Clean Beads, the same below) to the reaction tube for elution, and discard the supernatant; Add 300 μL of Wash Buffer1 used in conjunction with the magnetic beads to the reaction tube again for elution, and discard the supernatant; Then add 42 μL of elution buffer used in conjunction with the magnetic beads (i.e., elution buffer provided by Nanjing Novoprotein Science & Technology Co., Ltd. and used in conjunction with VAHTS DNA Clean Beads) to resuspend the magnetic beads. Remove from the magnetic stand and incubate at room temperature for 7 min, then transfer the reaction tube to the magnetic stand and let it stand for 5 min. After the magnetic beads are completely adsorbed, pipette 40 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube, and use the Equalbit 1×dsDNA HS Assay Kit to measure the DNA concentration in the supernatant.

[0179] 1.5. Sequencing

[0180] Refer to the CycloneSEQ sequencing kit instruction manual and use the CycloneSEQ sequencing kit and the MGI CycloneSEQ-WT02 gene sequencer to sequence the product obtained in step 1.4.

[0181] 2. Bioinformatics analysis

[0182] Use the Nanofilt software to perform quality control on the data downloaded from the sequencer, use the minimap software to align to the reference genome, use the samtools software to convert the file format, count the alignment results, use the bedtools software to generate a bed file, and use bamdst to count indicators such as coverage and sequencing depth.

[0183] 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, a genome coverage of greater than 95%, and a minimum detection limit of 1 copies / μL at a depth of 1000X.

[0184] Table 32 Nanopore sequencing results

[0185]

[0186] Example 3 (Clinical application of primer set)

[0187] The experimental samples of this example are: nucleic acid samples extracted from different types of samples in Table 3 (with numbers 20230230, 20210284, 20230403, 20230498-5). The acquisition method of the nucleic acid samples is as follows: Take 100 μL of the sample and use the nucleic acid extraction kit of Thermo Fisher Scientific to extract nucleic acid by the magnetic bead method using the nucleic acid automatic extractor KINGFISHER FLEX and MagMAX CORE Nucleic Acid Purification Kit. Use 90 μL of Elution Buffer to resuspend the nucleic acid to obtain 90 μL of nucleic acid solution; then take 5 μL of the nucleic acid solution and perform qPCR reaction through a PCR instrument to calculate the corresponding copy number based on the obtained CT value. Then perform the library construction experiment according to the following steps:

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

[0189] 2. Operate on the amplification product obtained in step 1 according to steps 3 to 5 of Example 1 to obtain the second-generation sequencing result, as shown in Table 33.

[0190] 3. Operate on the amplification product obtained in step 1 according to steps 1 to 2 of Example 2 to obtain the nanopore sequencing result, as shown in Table 33.

[0191] It can be seen from the test results of the clinical samples in Table 33 that the primer set of the present invention has good specificity, sensitivity, and a genome coverage of more than 95%. The lowest detection limit at a depth of 1000X can reach 1 copies / μL.

[0192] Table 33 Sequencing results of clinical samples

[0193]

[0194] Note in Table 33: The data volume of the second-generation sequencing is represented by the number of sequences; the data volume of the nanopore sequencing is represented by the number of bases.

[0195] 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. The library construction method has adaptability, can simplify manual operations, 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 method for constructing a whole genome library of porcine epidemic diarrhea virus and verified its feasibility through examples.

[0196] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words rather than limiting words. Modifications can be made to the present invention within the scope of the claims of the present invention as provided, and the present invention can 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 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 as 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 reagent includes the primer set described in claim 1.

3. The reagent according to claim 2, wherein The 27 pairs of primers with sequences shown as SEQ ID NO. 1 to SEQ ID NO. 54 are sequentially and alternately divided into primer set 1 and primer set 2. The reagent includes two groups of reagents, one group of reagents includes the primer set 1, and the other group of reagents includes the primer set 2.

4. A kit for amplifying the whole genome of porcine epidemic diarrhea virus, characterized in that, The kit includes the primer set described in claim 1 or the reagent described in claim 2 or 3.

5. The application of the primer set described in claim 1 or the reagent described in claim 2 or 3 or the kit described in claim 4 in the amplification of the whole genome of porcine epidemic diarrhea virus, and the application is for non-diagnostic purposes.

6. A method for constructing a library of the complete genome of porcine epidemic diarrhea virus for non-diagnostic purposes, characterized in that, The method includes performing multiplex PCR amplification using the primer set described in claim 1 or the reagent described in claim 2 or 3 or the kit described in 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) Reverse transcribe the nucleic acids extracted in step (1) to obtain cDNA; (3) Perform multiplex PCR amplification on the cDNA obtained by reverse transcription in step (2) using the primer set described in claim 1 or the reagent described in claim 2 or 3 or the kit described in claim 4; (4) Construct a sequencing library; (5) Sequence on a machine; (6) Bioinformatics analysis.

8. The method according to claim 7, wherein The samples in step (1) include one or more of feces, intestinal tissues, and anal swabs; 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 by amplification in step (3) is between 1000 and 1400 bp; And / or, the amplification program in step (3) is: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 65°C for 30 s, extension at 72°C for 2 min, for a total of 35 cycles; extension at 72°C for 2 min; And / or, the reaction reagents for amplification in step (3) include VAHTS Pathogen DNA Multiplex PCR Mix, cDNA, ddH2O, and the primer set described in claim 1; And / or, step (3) further includes purifying the amplification product obtained by amplification; And / or, the construction of the sequencing library in step (4) 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 27 pairs of primers with sequences shown as SEQ ID NO. 1 to SEQ ID NO. 54 are sequentially and alternately divided into primer set 1 and primer set 2. The amplification in step (3) is carried out in two groups, and the nucleic acids extracted in step (1) are subjected to multiplex PCR amplification using the primer set 1 and the primer set 2 respectively, and then the amplification products are combined.

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

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

12. The method according to claim 11, wherein When the construction of the sequencing library in step (4) is carried out on a next-generation sequencing platform, the steps of constructing the sequencing library further include purifying the product obtained in the adapter ligation step and / or purifying the product obtained in the enzymatic digestion step; When the construction of the sequencing library in step (4) is carried out on a nanopore sequencing platform, the steps of constructing the sequencing library further include purifying the product obtained in the end repair and 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.

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

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