Primer set for detecting porcine hev whole genome, kit comprising the primer set and application

Abstract

The application discloses a primer set for detecting a whole genome of a pig HEV, a kit comprising the primer set and application. The primer set comprises nucleotide sequences as shown in SEQ ID NO. 1-62, the primer set is designed for amplification fragments of 450 bp of a whole gene of the pig HEV and 650 bp of a non-conserved region, is divided into two sets of shingle degenerate primers, comprises five primer groups of A, B, C, D and E, and five amplification primer pools are constructed by using the five primer groups, so that the primer set can cover the complete genome of different strain types of the pig HEV. The application also establishes a method for sequencing the whole genome of the pig hepatitis E virus by using multiplex targeted RT-PCR and nanopore sequencing based on the primer set. The method is rapid, sensitive and specific, can be used for rapidly obtaining the whole genome information of the pig hepatitis E virus in a sample, and provides a reliable technical means for tracing the pig hepatitis E virus and accurately determining the genotype.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal quarantine, in particular to a primer set for detecting a whole genome of a porcine HEV, a kit comprising the primer set and an application thereof. BACKGROUND

[0002] Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus with a genome length of about 7.2-7.4 kb and a virus particle diameter of about 27-34 nm. It mainly contains three open reading frames (ORF1, ORF2 and ORF3), and ORF4 only exists in HEV-1. The virus belongs to the Orthohepevirus genus of the Hepeviridae family of Orthohepevirus and can infect multiple hosts. It has been confirmed as an important zoonotic pathogen.

[0003] HEV can be currently divided into eight genotypes, of which HEV-1 to HEV-4 can infect humans. HEV-1 and HEV-2 only infect humans; while HEV-3 and HEV-4 are zoonotic, with pigs as the main host, and can be transmitted to humans through infected pigs and their products. Studies have confirmed that sHEV is highly homologous to the human HEV genome and has the ability to cross-species transmission, including from pigs to non-human primates (such as rhesus monkeys and chimpanzees), and vice versa from humans to pigs. This bidirectional transmission phenomenon highlights that pigs can serve as a reservoir host for HEV-3 and HEV-4 genotypes, posing a persistent risk to public health.

[0004] The transmission routes of HEV mainly include fecal-oral route, consumption of undercooked meat products, blood transfusion transmission and vertical transmission. Among them, consumption of pork products that have not been thoroughly heated is one of the main routes of sHEV transmission to humans. It is worth noting that the HEV RNA positive rate and viremia level in the feces of 2-4 month-old piglets are high, and although the seroprevalence rate decreases with age, it can still continuously excrete viruses through feces. In addition, the virus can also exist in the bile, liver and lymph nodes of pigs for a long time, further increasing the risk of transmission.

[0005] In terms of detection technology, the commonly used HEV nucleic acid analysis methods currently include RT-PCR combined with Sanger sequencing, real-time fluorescent RT-PCR, etc., and the sample types used are mostly serum, feces or liver tissue. However, as a single-stranded RNA virus, HEV is easily degraded in samples, and the actual detection is often limited by factors such as low viral load, strict sample storage conditions and large sequence variation among different strains. The existing methods generally have problems such as low throughput, poor coverage integrity, long time consumption and insufficient sensitivity for low-load samples. In particular, it is worth noting that there is currently a lack of a targeted enrichment scheme that can efficiently amplify the whole genome of sHEV and is compatible with long-read sequencing (such as nanopore sequencing), which severely limits the application in genotyping, traceability analysis and variation monitoring, etc.

[0006] Therefore, it is urgent to develop a high-throughput detection method that can efficiently enrich the whole genome of sHEV, overcome sequence variation interference, be suitable for low-load clinical samples, and be combined with third-generation sequencing technology, so as to realize rapid and accurate whole genome determination and molecular traceability. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a primer set for detecting the whole genome of porcine HEV, a kit comprising the primer set and an application. Based on sHEV of genotype 3 and genotype 4 from different sources, i.e. porcine HEV-3 and HEV-4, the present application designs a redundant primer set for capturing and enriching sHEV according to the target region, and constructs a method for nanopore sequencing of the whole genome of porcine hepatitis E virus, which has high sensitivity and specificity. The redundant primer set has application prospects in sHEV whole genome amplification, library construction and sequencing traceability.

[0008] To achieve the above purpose, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a primer set for detecting the whole genome of porcine HEV, comprising the nucleotide sequences shown as SEQ ID NO. 1-62.

[0010] In a second aspect, the present application provides a reagent for detecting the whole genome of porcine HEV, comprising the primer set of the first aspect.

[0011] In a third aspect, the present application provides a kit for detecting the whole genome of porcine HEV, comprising the reagent of the second aspect.

[0012] In a fourth aspect, the present application provides a PCR method for enriching the whole genome of porcine HEV, comprising using the primer set of the first aspect as PCR amplification primers.

[0013] In a fifth aspect, the present application provides a method for detecting the genome of porcine HEV, comprising:

[0014] (1) adopting the PCR method of the fourth aspect to target amplify the whole genome of pig HEV to obtain an amplification product;

[0015] (2) constructing a sequencing library for the amplification product of (1);

[0016] (3) nanopore sequencing of the sequencing library of (2);

[0017] (4) genotype typing and molecular tracing analysis according to the sequencing results of (3).

[0018] Compared with the prior art, at least the following beneficial effects are achieved:

[0019] 1. The primer set designed in the present application has excellent specificity and broad spectrum, and high analysis sensitivity for pig hepatitis E virus whole genome nucleic acid enrichment and resequencing. The primer set has application prospects in sHEV whole genome amplification, library construction and sequencing tracing.

[0020] 2. The pig hepatitis E virus whole genome targeted nanopore sequencing method established based on the above-mentioned primer set has the following advantages: (1) broad spectrum: pig hepatitis E virus mainly includes genotype 3 and genotype 4, and there are multiple variant strains within the same genotype. The present method is based on a large number of strain gene sequence alignment, and redundant degenerate primers are designed for non-conserved regions, which can effectively capture the viral gene sequences of different genotypes and different strains, and can effectively avoid the problem of primer failure caused by strain variation; (2) fast: the method can directly construct a library and perform nanopore sequencing on the RT-PCR target amplification product, and the targeted sequence is highly specific, so the data generated in a short time during sequencing can obtain the viral genome sequence, and the spliced sequence can be used for viral gene typing and tracing analysis; (3) high sensitivity: the sequencing sensitivity can reach 10 copies / μL; (4) high specificity: the method can specifically target and enrich pig hepatitis E virus genomic RNA; (5) wide range of applicable samples: the redundant primer pool design and small amplification fragments make the amplification efficiency high, and even if the viral genomic RNA in the sample is not complete, it can also be effectively amplified and enriched.

[0021] 3. The library construction method of the present application is suitable for the third generation nanopore sequencing platform, and has high automation. Even in a sample with a low virus content of 10 copies / μL, more than 85% of the viral sequences can be obtained, and the sequencing depth can reach more than 10-100 times. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The design strategy schematic diagram of the redundant shingled primer set provided by the present application for targeting amplification and enrichment of pig hepatitis E virus genome.

[0023] Figure 2 Technical route of porcine hepatitis E virus whole genome sequencing by using the redundant primer set provided in the present application on a nanopore sequencing platform.

[0024] Figure 3 Capillary electrophoresis chart of RT-PCR product in Example 1 of the present application.

[0025] Figure 4 Capillary electrophoresis chart of RT-PCR product in Example 3 of the present application.

[0026] Figure 5 Microorganism identification Sankey chart of sequencing result provided in Example 3 of the present application.

[0027] Figure 6 Porcine hepatitis E virus whole genome coverage provided in Example 3 of the present application.

[0028] Figure 7 Evolution analysis result of porcine hepatitis E virus whole genome sequence in the sample in Example 3 of the present application is shown. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0030] The materials used in the following examples are not limited to the following list, and other similar materials can be used instead. If the specific conditions of the instruments are not specified, the instruments are used according to the conventional conditions or the conditions recommended by the manufacturers. Those skilled in the art should master the relevant knowledge of using conventional materials and instruments.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described in this application belongs. Before the present application is described in detail, the following definitions are provided to better understand the present application.

[0032] In order to better understand the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or ratios and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present teachings. At the very least, each numerical parameter should at least be construed in light of the number of significant digits it contains and by applying ordinary rounding techniques.

[0033] In the context of the present application, many embodiments use the expressions "comprising", "including". The expression "comprising" or "including" can be understood in the broadest sense as an open-ended expression indicating that not only the listed elements, components, assemblies, method steps, etc. are included, but also other elements, components, assemblies, method steps, etc. are included. In addition, in this text, the expression "comprising" or "including" can also be understood in some cases as a closed expression, indicating that only the elements, components, assemblies, method steps listed after the expression are included, and no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of".

[0034] In the text of the present application, there are several different expressions for primer sets, such as the expressions "primer sets A, B, C, D, E", "Waston primer set", "redundant primer set", "redundant Waston primer set", "degenerate primer set", etc. Those skilled in the art should not have difficulty understanding that these expressions are synonymous and refer to all or part of the SEQ ID NO. 1-62 primers provided in the present application.

[0035] To solve the problem of detecting the genome of porcine hepatitis E virus in the prior art, the present inventors compared the genome sequence of porcine hepatitis E virus, adopted a redundant primer strategy, designed two sets of Waston degenerate primer sets for about 450bp and about 650bp amplification fragments respectively for the whole gene and non-conserved region, which can ensure that the primer set can cover the complete genome of different strains of virus, and there is an overlapping region of about 100bp between adjacent primer pairs. The adjacent primer pairs are respectively set as 5 different primer pools, and to ensure sensitivity, there are at most 7 primer pairs in each primer pool. Then, 5 different primer pools are used for amplification and the amplification products are combined, which can effectively avoid non-specific hybridization and amplification of primers and improve the amplification efficiency.

[0036] Based on this, the embodiments of the present application provide a primer set for detecting the whole genome of porcine HEV, which comprises the nucleotide sequences shown as SEQ ID NO. 1-62.

[0037] In the above primer design rules, the length of the primer is 19-29 bases, the GC content is 50%-60%, there is no secondary and repetitive structure in the primer, there is no complementary sequence between and within the primers, and the melting temperature (Tm value) between the primers is less than 5℃.

[0038] In actual use, the above 62 primer sequences are divided into 5 groups, i.e. primer sets A, B, C, D, E, as shown in Table 1. Figure 1As shown, the inventors further divided the 5 primer sets into two sets of imbricate degenerate primer sets, wherein primer sets A, B and C are grouped as one set for amplifying the whole gene region of porcine hepatitis E virus, and primer sets D and E are grouped as another set for amplifying the non-conserved region of porcine hepatitis E virus, so as to realize efficient enrichment of the whole gene targeting of porcine hepatitis E virus and subsequent genotyping. The sequences and grouping information of the above 62 primers are shown in Table 1 below:

[0039] Table 1 sHEV whole genome targeted capture enrichment primer set

[0040]

[0041]

[0042] In the above primer sequences, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the degenerate base R represents A / G, Y represents C / T, M represents A / C, K represents G / T, W represents A / T, H represents A / C / T, V represents A / C / G, S represents G / C, and the letter I represents inosine (also known as inosine).

[0043] Based on this, the embodiments of the present application provide a reagent for detecting the whole genome of porcine HEV, which comprises the primer set of the nucleotide sequences shown in the foregoing SEQ ID NO. 1-62.

[0044] Based on this, the embodiments of the present application also provide a kit for detecting the whole genome of porcine HEV, which comprises the above-mentioned reagent.

[0045] In certain embodiments, the kit further comprises a multiplex RT-PCR reaction solution, an enzyme mixture, and nuclease-free water. These reagents are required for the re-targeting RT-PCR amplification and enrichment reaction of the porcine HEV gene; the multiplex RT-PCR reaction solution can comprise an RT-PCR buffer, MgCl2, dNTP, a reverse transcriptase, an RNase inhibitor, and a Taq DNA polymerase.

[0046] Based on this, the embodiments of the present application provide a PCR method for enriching the whole genome of porcine HEV, which comprises using the foregoing primer set as PCR amplification primers.

[0047] In the above PCR method, in addition to using the primer set of the nucleotide sequences shown in SEQ ID NO. 1-62 provided by the present application as amplification primers, other aspects in the PCR amplification are not specifically set, and the amplification purpose is used as the criterion.

[0048] In certain embodiments, the specific steps of the PCR method are as follows:

[0049] collecting a sample, extracting nucleic acid in the sample;

[0050] using the extracted nucleic acid as a template, using the aforementioned primer groups A, B, C, D, and E for multiplex RT-PCR amplification reaction, respectively, to obtain five groups of amplification products;

[0051] mixing the five groups of amplification products in equal volumes to obtain the final RT-PCR mixed amplification product of the whole genome of porcine HEV.

[0052] In some embodiments, the method for extracting nucleic acid in the sample is a magnetic bead method or a column method.

[0053] In some embodiments, the sample is derived from at least one of pig feces, bile, liver, lymph node, or blood.

[0054] In some preferred embodiments, the amplification system of the PCR method is a 20 μL system:

[0055] 2xHifair V MP Buffer 10 μL, Hifair V Enzyme Mix 1.5 μL, primer pool A or B or C or D or E 0.2 μmol / L (final concentration of each primer), nucleic acid template 5 μL, supplemented with nuclease-free water to 20 μL;

[0056] The reaction conditions are as follows:

[0057] 50℃ / 15min;

[0058] 95℃ / 5min;

[0059] 95℃ / 15s, 55℃ / 30s, 60℃ / 90s, 40 cycles.

[0060] Based on this, the embodiments of the present application provide a detection method for the genome of porcine HEV, comprising:

[0061] (1) using the aforementioned PCR method to target amplify the whole genome of porcine HEV to obtain a mixed amplification product;

[0062] (2) constructing a sequencing library for the amplification product of (1);

[0063] (3) performing nanopore sequencing on the sequencing library of (2);

[0064] (4) performing genotyping and molecular tracing analysis according to the sequencing results of (3).

[0065] Figure 2The technical roadmap for detecting the porcine HEV genome using the aforementioned primer groups A, B, C, D, and E on a nanopore sequencing platform is shown, starting from virus nucleic acid extraction, through PCR amplification, data analysis, to library sequencing, to achieve detection and analysis of viral genes. The following will be combined with Figure 2 The detection method is described in detail as follows:

[0066] (1) Virus genomic DNA / RNA extraction: isolate viral nucleic acid from the sample to prepare for subsequent detection;

[0067] (2) Construction of multiplex RT-PCR reaction system: mix the extracted nucleic acid with "multiplex RT-PCR Mix" (containing enzymes, primers, etc.), primer pool (different primers such as A-E), and specifically amplify the viral target gene fragments through the "multiplex RT-PCR target capture" instrument;

[0068] (3) Amplification product processing and analysis: after mixing the amplification products, "product purification and quantification" is performed, and "sequence splicing and gene clustering analysis" is performed using software to preliminarily analyze the gene information;

[0069] (4) Library construction and sequencing: construct a library by adding "native barcode" and "native adapter", and finally use a sequencer to "sequence determination" to obtain viral gene sequence data for virus identification, typing, and variation analysis research.

[0070] The technical solutions and technical effects achieved by the present application will be described in more detail through more specific examples.

[0071] Example 1: Establishment of a porcine hepatitis E virus whole genome targeted nanopore sequencing method

[0072] 1. Primer probe composition for porcine hepatitis E virus whole genome targeted nanopore sequencing

[0073] In the present application, the whole gene of porcine hepatitis E virus is selected as the target region, and on the basis of multiple sequence alignment, a redundant shingled primer composition is designed, and the redundant primer group amplification strategy is shown in Figure 1 The length of the primers is 19-29 bases, the GC content is 50%-60%, there is no secondary structure and repetition in the primers, there is no complementary sequence between and within the primers, and the melting temperature (Tm value) between the primers differs by less than 5°C. The final designed redundant primer group includes single-stranded DNA molecules represented by SEQ ID NO. 1-62, and the 62 primers are divided into five groups A, B, C, D, and E. The nucleotide sequences of the primers and the grouping information are shown in Table 1.

[0074] 2. Method for targeted amplification and enrichment of porcine hepatitis E virus whole genome

[0075] The method for using multiplex RT-PCR for the whole genome of porcine hepatitis E virus is as follows: the pretreatment of the sample to be tested and the extraction of nucleic acid, using the extracted nucleic acid as a template, preparing A, B, C, D and E primer pools respectively with the five aforementioned primers, and performing multiplex RT-PCR amplification, and using the RT-PCR product after mixing and purification for nanopore sequencing, which specifically includes the following steps:

[0076] 2.1 Pretreatment of the sample to be tested and extraction of nucleic acid

[0077] For the sample to be tested, the pretreatment and extraction of nucleic acid are performed according to the following method:

[0078] For liquid samples such as fecal swabs, bile and blood, the sample can be directly taken and used to extract total nucleic acid with a viral genomic DNA / RNA extraction kit (Tiangen viral genomic DNA / RNA extraction kit, DP315) according to the instructions; for liver, lymph node and other samples, the sample is ground and made into a 20% tissue suspension with PBS, centrifuged at 3000 r / min for 5 min, and the supernatant is taken for viral nucleic acid extraction; effective nucleic acid extraction methods can be used, and various commercial viral nucleic acid extraction kits or automatic nucleic acid extraction instruments and matching reagents can also be used for viral nucleic acid extraction; the extracted nucleic acid must be detected within 2 h or stored at -70°C or below.

[0079] In the construction of the detection method, the positive pig fecal swab is used as a sample to extract viral nucleic acid.

[0080] 2.2 Targeted multiplex RT-PCR amplification

[0081] The pig fecal swab nucleic acid obtained in step 2.1 is used as a template, and A, B, C, D and E primer pools are used for multiplex RT-PCR amplification to obtain various RT-PCR products, and the targeted multiplex RT-PCR amplification reaction system is shown in Table 2:

[0082] Table 2 Multiplex RT-PCR amplification reaction system

[0083] Components Volume or final concentration 2x Hifair VMP Buffer 10 μL Hifair V Enzyme Mix 1.5 μL Primer Pool A or B or C or D or E 0.2 μmol / L (final concentration per primer) Nucleic acid template 5 μL Supplement DEPC (nuclease-free) water 20 μL

[0084] Among them, 2xHifair V MP Buffer and Hifair V Enzyme Mix are purchased from YEASEN company; primers are synthesized by Shanghai Shengong Biological Engineering Co., Ltd.; primer pools A, B, C, D and E are prepared by mixing according to Table 2; DEPC water is distilled twice with pure water, DEPC is added to a final concentration of 0.1%, and stirred at 37°C for 12h, 1.034x10 5 Pa high pressure steam sterilization for 15 minutes.

[0085] The reaction procedure of the targeted amplification multiplex RT-PCR is shown in Table 3 below:

[0086] Table 3

[0087]

[0088] The RT-PCR product electrophoretogram is shown in Figure 3 As can be seen from the figure, the RT-PCR amplification is successful, and the RT-PCR product is stored after 0.8x magnetic bead purification.

[0089] 3. RT-PCR product for nanopore sequencing

[0090] According to the operation instruction of the nanopore sequencing related kit (including bar code connection sequencing kit SQK-NBD114.24; R10 sequencing chip, FLO-MIN114, NANOPORE; connection method sequencing multi-sample DNA library construction auxiliary reagent kit, BAIYI), the specific steps include the following steps:

[0091] 3.1 RT-PCR product purification:

[0092] ①Balance the AMPure XP magnetic beads in the kit to room temperature, shake and suspend, take 40 μL magnetic beads and add to 50 μL mixed RT-PCR product, mix by flicking, and incubate at room temperature for 5 minutes; ②After short centrifugation, transfer the liquid to a new 1.5 mL centrifuge tube, place it on the magnetic stand, and wait for the liquid to clarify; ③Remove the supernatant on the magnetic stand, add 180 μL of freshly prepared 80% ethanol along the wall of the tube to gently wash the magnetic beads, then discard the liquid, and then repeat the washing again; ④After short centrifugation, place the centrifuge tube back on the magnetic stand, remove the residual liquid; open the cover and dry naturally for 30 seconds; ⑤Add 27 μL of nuclease-free water to resuspend the magnetic beads for nucleic acid elution, and place it at room temperature for 2 min, then centrifuge briefly and place it on the magnetic stand until the liquid is clarified; take 1 μL to measure the concentration with Qubit dsDNA HS Assay Kit, then take 25 μL for the next step of end repair.

[0093] 3.2 End repair

[0094] 1. Add 25 μΐ of purified RT-PCR product to a 0.2 mL PCR tube; 2. Add reagent components of the kit according to Table 4 below, mix by flicking and centrifuge briefly; 3. Incubate at 20°C for 5 min on a PCR machine, then at 65°C for 5 min, and remove; 4. Purify using equal volume of AMPure XP beads according to Step 3.1, elute the beads after the final ethanol wash with 15 μΐ of nuclease-free water; take 1 μΐ for concentration measurement using Qubit dsDNA HS Assay Kit, and take 13 μΐ for the next step of adding native barcode.

[0095] Table 4 End repair reaction system

[0096] Components Volume (μL) RT-PCR product 25 End Repair Buffer (ERB) 3.5 End Repair Enzyme (ERE) 1.5 Total volume 30

[0097] 3.3 Adding native barcode

[0098] 1. Add reagent components of the kit according to Table 5 below, mix by flicking and centrifuge briefly; 2. Mix by flicking for about 10 times, centrifuge briefly, and incubate at 18-25°C for 10 min; 3. Purify using equal volume of AMPure XP beads according to Step 3.1, elute the beads after the final ethanol wash with 15 μΐ of nuclease-free water; take 1 μΐ for concentration measurement using Qubit dsDNA HS Assay Kit, and take 13 μΐ for the next step of library construction.

[0099] Table 5 System for adding native barcode

[0100] Components Volume (μL) End-repaired DNA 13 Native barcode 2 Ligation Master Mix (LMM) 14.5 Ligation Enhancer Buffer (LEB) 0.5 Total volume 30

[0101] 3.4 Library construction

[0102] ① The purified DNA with connected barcodes was diluted to 51 μL with nuclease-free water, added to a new 1.5 mL centrifuge tube, and the system was prepared according to Table 6 below to connect the sequencing adapter and construct the sequencing library; ② gently mix for about 10 times, centrifuge briefly, and incubate at 18-25 °C for 10 min; ③ add 40 μL AMPure XP magnetic beads, mix by tapping the tube wall, and incubate at room temperature for 5 min; ④ centrifuge briefly and place on the magnetic stand, and after the liquid is clarified, the supernatant is aspirated; ⑤ remove the centrifuge tube, gently wash the magnetic beads with 150 μL SFB, centrifuge briefly, and place on the magnetic stand, and after the liquid is clarified, the supernatant is aspirated. Then repeat the washing once; ⑥ centrifuge briefly and place the centrifuge tube back on the magnetic stand, aspirate the residual liquid, and open the cap to dry naturally for 30 seconds; add 14 μL EB elution solution, remove the centrifuge tube from the magnetic stand, mix gently, and incubate at room temperature for 10 min; place the centrifuge tube on the magnetic stand until the elution solution is transparent and colorless; aspirate the elution solution into a new 1.5 mL centrifuge tube, and take 1 μL to measure the concentration with Qubit dsDNA HS Assay Kit.

[0103] Table 6 Library construction system preparation

[0104] Components Volume (μL) Barcoded DNA 51 Native Adapter 5 Adapter Ligation Buffer (ALB) 16 Adapter Ligation Enzyme (ALE) 8 Total volume 80

[0105] 3.5 Third-generation nanopore sequencing

[0106] ① Thaw Sequencing Buffer (SB), Library Beads (LIB), FlowCell Tether (FCT), Flow Cell Flush (FCF) in the kit at room temperature, vortex mix, centrifuge briefly, and keep on ice; ② Add 30 μL FCT to 1170 μL FCF, vortex mix; ③ After the sequencing chip is equilibrated at room temperature for 10 min, rotate it clockwise by 90° to open the Priming port, adjust a 1 mL pipette to 200 μL, and vertically touch the Priming port hole to rotate slowly to 230 μL in the air bubble in the pipeline. At this time, there is a small amount of liquid at the tip of the gun, indicating that the pipeline bubble is discharged; ④ Use a 1 mL pipette to take 800 μL of FCF with FCT, and push it into the pipeline at a constant speed from the Priming port hole by rotating the pipette clockwise. Note that a small amount of liquid is retained at the tip of the gun. Equilibrate the chip at room temperature for another 5 min; ⑤ After the chip is equilibrated, open the SpotON sample hole by tapping it outward; ⑥ Add each reagent in turn according to Table 7 below to prepare the sequencing sample system; ⑦ After blowing and sucking the sample system, add it dropwise to the SpotON sample hole; ⑧ Close the SpotON sample hole first, then close the Priming port, and then suck the waste liquid from the waste hole. Cover the chip with a light shielding sticker, and load it into the GridION for sequencing.

[0107] Table 7 Preparation of sequencing sample system

[0108] Components Volume (μL) Sequencing Buffer (SB) 37.5 Library magnetic beads (LIB, mix well before pipetting) 25.5 Purified DNA library 12 Total volume 75

[0109] 3.6 Sequencing data analysis

[0110] The generated sequence data is filtered to remove sequencing adapters and barcodes, and full-length sequence fragments with a length of more than 250 bp are retained to ensure the integrity and applicability of the data. The sequencing data is imported into a microbial identification database for sequence assembly and identification to determine the sequencing depth and coverage. The assembled porcine hepatitis E virus full sequence is subjected to evolutionary analysis with different genotype sequences published in GenBank to determine the viral evolutionary lineage, genotype, and source by evolutionary clustering.

[0111] Example 2 Sensitivity evaluation of porcine hepatitis E virus full genome targeted nanopore sequencing method

[0112] A pig positive fecal swab is used as a sample to be tested to evaluate the sensitivity of the porcine hepatitis E virus targeted nanopore sequencing method, and the specific method is as follows:

[0113] The viral nucleic acid was extracted using the Tian Gen viral genomic DNA / RNA extraction kit (DP315). After the viral sampling tube was shaken thoroughly, it was allowed to stand for 1 min. 200 μL of the sample was then taken and the viral nucleic acid was extracted according to the instructions. After the nucleic acid was extracted, the method reported by Jothikumar was used in combination with the known copy number of in vitro transcribed RNA as an external standard quality control to determine the HEV RNA copy number in the sample. The RNA concentration in the sample was adjusted to 1-1000 copies / μL.

[0114] The foregoing A, B, C, D, and E primer pools were used to perform targeted RT-PCR amplification with 4 groups of different concentrations of RNA as templates, and the viral nucleic acid extracted from negative pig fecal swabs was used as a negative control. After the mixed products of the 4 groups of targeted RT-PCR were purified, the nucleic acid concentration was determined using Qubit, and then sequencing was performed according to the nanopore sequencing method of Example 1. The results are shown in Table 8:

[0115] Table 8 Sequencing sensitivity evaluation

[0116]

[0117] As can be seen from the results in Table 8, when the RNA concentration is 10 copies / μL, the genome sequence coverage is about 85%. This indicates that the sensitivity of the porcine hepatitis E virus targeted genomic nanopore sequencing method established by the present application is 10 copies / μL.

[0118] Example 3 Tracing of porcine hepatitis E virus positive samples of imported breeding pigs by targeted nanopore genomic sequencing

[0119] The sequencing method of Example 1 was used to perform whole genome targeted nanopore resequencing of porcine hepatitis E virus positive samples of imported breeding pigs. The specific method is as follows:

[0120] 1. Sample collection and nucleic acid extraction

[0121] The sample was a fecal swab sample from an imported live pig, and the viral nucleic acid was extracted using the Tian Gen viral genomic DNA / RNA extraction kit (DP315). The tube with the swab sample was shaken thoroughly, and then allowed to stand for 1 min. 200 μL of the sample was then taken, and the viral nucleic acid was extracted according to the instructions of the extraction kit.

[0122] 2. Targeted RT-PCR amplification using the viral nucleic acid extracted in step 1 as a template

[0123] The foregoing primer groups A, B, C, D, and E were used to prepare the corresponding redundant primer pools, and the hepatitis E virus positive pig fecal swab nucleic acid was used as a template to perform multiplex RT-PCR. The viral nucleic acid extracted from negative pig fecal swabs was used as a negative control.

[0124] The results of the multiplex RT-PCR electrophoresis are shown in Figure 4 The results preliminarily show that the viral nucleic acid can be effectively amplified in the primer pool.

[0125] 3. Nanopore sequencing

[0126] The RT-PCR products were subjected to library construction and nanopore sequencing according to Example 1.

[0127] 4. Analysis of results

[0128] After 2h of nanopore sequencing, data analysis was performed. The generated sequence data was subjected to filtering and clustering analysis, and then the measured data was imported into a microbial analysis database for microbial species identification analysis, and a Sankey diagram was drawn, as shown in Figure 5 .

[0129] Figure 4 The results show that a large proportion of porcine hepatitis E virus sequences exist in the viral data in the sequences.

[0130] The porcine hepatitis E virus gene sequence was extracted from the data, and a reference sequence was selected for parametric sequence assembly, and the HEV full gene sequence in the pig fecal swab sample was obtained, with a coverage of 99.6% and an average depth of 8959.39, as shown in Figure 6 . Then, the gene sequence was subjected to sequence alignment with the sequences of different genotypes of representative strains in GenBank, and a phylogenetic tree was drawn using the UPGMA model using the Mega (version 12.0) software, with a bootstrap of 1000, as shown in Figure 7 . From the figure, it can be seen that the measured sequence is classified into an evolutionary branch with the 3e genotype, indicating that the viral nucleic acid in the sample is the 3e gene subtype of HEV-3 genotype.

[0131] The above has described the present application in detail, and the principles and implementation modes of the present application have been described by applying specific examples. The above description of the examples is only for the purpose of helping to understand the present application and the core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1.A primer set for detecting a whole genome of porcine HEV, the nucleotide sequences of which are shown in SEQ ID NO.1-62. 2.A reagent comprising the primer set for detecting a whole genome of porcine HEV according to claim 1. 3.A kit comprising the reagent according to claim 2. 4.The kit according to claim 3, further comprising a multiplex RT-PCR reaction solution, an enzyme mixture, and nuclease-free water, wherein the multiplex RT-PCR reaction solution can comprise an RT-PCR buffer, MgCl 2, dNTP, a reverse transcriptase, an RNase inhibitor, and a Taq DNA polymerase. 5.A PCR method for enriching a whole genome of porcine HEV for non-diagnostic purposes, comprising the following steps: collecting a sample and extracting nucleic acids in the sample; using the extracted nucleic acids as templates, performing a multiplex RT-PCR amplification reaction using primer sets A, B, C, D, and E to obtain five groups of amplification products, wherein the primer set A comprises the primer sequences of SEQ ID NO.1, 2, 8, 9, 15, 16, 21, 22, 27, 28, 33, 34, 39, and 40; the primer set B comprises the primer sequences of SEQ ID NO.3, 4, 5, 10, 11, 17, 18, 23, 24, 29, 30, 35, 36, 41, and 42; the primer set C comprises the primer sequences of SEQ ID NO.6, 7, 12, 13, 14, 19, 20, 25, 26, 31, 32, 37, 38, 43, and 44; the primer set D comprises the primer sequences of SEQ ID NO.45, 46, 49, 50, 53, 54, 57, 58, 61, and 62; and the primer set E comprises the primer sequences of SEQ ID NO.47, 48, 51, 52, 55, 56, 59, and 60; mixing the five groups of amplification products in equal volumes to obtain a final RT-PCR mixed amplification product of the whole genome of porcine HEV. 6.The PCR method according to claim 5, wherein the method for extracting nucleic acids in the sample is a magnetic bead method or a column method. 7.The PCR method according to claim 5, wherein the sample nucleic acids are derived from at least one of pig feces, bile, liver, lymph nodes, or blood. 8.The PCR method according to claim 5, wherein the amplification system of the PCR method is a 20 μL system: 2×Hifair V MP Buffer 10 μL, Hifair V Enzyme Mix 1.5 μL, each primer in primer sets A, B, C, D, or E at a final concentration of 0.2 μmol / L, nucleic acid templates 5 μL, and supplemented with nuclease-free water to 20 μL; the reaction conditions are as follows: 50 ℃ / 15 min; 95 ℃ / 5 min; 95 ℃ / 15 s, 55 ℃ / 30 s, 60 ℃ / 90 s, 40 cycles. 9.A method for obtaining a whole genome sequence of porcine HEV for non-diagnostic purposes, comprising the following steps: (1) The whole genome of porcine HEV is targeted amplified using the PCR method described in any one of claims 5 to 8 to obtain mixed amplification products; (2) Construct a sequencing library from the amplification products of (1); (3) Perform nanopore sequencing on the sequencing library from (2); (4) Genotyping and molecular origin analysis were performed based on the sequencing results of (3).

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