Porcine epidemic diarrhea virus whole genome amplification primer and method based on nanopore sequencing

Abstract

The invention provides an amplification primer and method for a porcine epidemic diarrhea virus whole genome. The detection primer group for the porcine epidemic diarrhea virus comprises 60 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-120. The detection primer group provided by the invention can realize relatively uniform coverage of the porcine epidemic diarrhea virus, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the porcine epidemic diarrhea virus whole genome is simple, convenient and easy to operate and good in amplification effect, nanopore sequencing has the advantages of sequencing and real-time analysis at the same time, the detection time can be greatly shortened, infection of the porcine epidemic diarrhea virus can be rapidly identified and diagnosed, meanwhile, the whole genome sequence of the porcine epidemic diarrhea virus is obtained, and the application prospect is wide. The obtained genome sequence can provide a scientific basis for virus traceability, pathogenic variation tracking, novel strain identification, early warning and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of animal virus detection, and in particular to a primer and method for amplifying the whole genome of porcine epidemic diarrhea virus based on nanopore sequencing. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, contagious gastrointestinal disease caused by porcine epidemic diarrhea virus (PEDV). Clinically, it can cause severe diarrhea, vomiting and dehydration in pigs, and the mortality rate of piglets under ten days old is as high as 100%, causing huge economic losses and production difficulties to the pig farming industry. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-strand RNA virus belonging to the alpha-coronavirus; it is divided into two subtypes, G1 and G2. The first discovered and popular strain was the G1 subtype represented by PEDV CV777. However, since October 2010, a highly pathogenic G2b variant has begun to break out and spread, causing serious damage to the pig farming industry.

[0003] In production, PED can infect pigs of all ages and cause disease. It is most harmful to piglets under 10 days old, with a mortality rate of up to 100%. This disease occurs in all seasons, but is more common in winter and spring. PEDV strains mutate rapidly. Rapid differential diagnosis of PEDV infection and obtaining its genomic information can provide a basis for early detection, early control, and traceability of the virus, and minimize the economic losses it brings to the pig industry. At present, the main methods for virus detection are virus isolation and culture, immunological detection, and nucleic acid detection, but none of the above three methods can obtain the complete genome sequence of the virus and cannot analyze the characteristics of the viral genome. Although both first-generation and second-generation sequencing can obtain the full length of the viral genome, first-generation sequencing requires independent sequencing of the amplified fragments, which has a relatively low throughput and is time-consuming and labor-intensive. Although second-generation sequencing has a higher throughput, it is usually sequenced simultaneously with the host genome, and short sequences need to be assembled, which is prone to errors. In addition, since the second-generation sequencing platform usually has too high a throughput, it is not suitable for real-time detection. Nanopore sequencing has the characteristics of real-time, portability, long read length, and high throughput, and can achieve real-time rapid detection on site; however, there is currently no sequencing technology for PEDV designed for the advantages of nanopore sequencing. Therefore, there is still an urgent need for a whole genome sequencing technology for porcine epidemic diarrhea virus based on third-generation nanopore sequencing. Summary of the invention

[0004] The present invention designs specific primers for PCR amplification of the whole genome of PEDV, and further provides a real-time detection method for PEDV using nanopore sequencing, which gives full play to the advantages of nanopore sequencing in pathogen detection, such as real-time, portability, long read length, and high throughput. The detection method can quickly differentiate and diagnose the infection of different genotypes of PEDV, and obtain its whole genome sequence at the same time. The obtained whole genome sequence can provide information for pathogen variation monitoring and molecular epidemiological research.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides a detection primer set for porcine epidemic diarrhea virus, the detection primer set comprising 60 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 120.

[0007] On the other hand, the present invention provides use of the above detection primer set in preparing a product for detecting porcine epidemic diarrhea virus.

[0008] Furthermore, the product includes a reagent or a kit.

[0009] On the other hand, the present invention provides a detection reagent for porcine epidemic diarrhea virus, comprising the above-mentioned detection primer set.

[0010] Furthermore, the detection reagent includes a first primer set and a second primer set, wherein the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.61 to 120.

[0011] On the other hand, the present invention provides a detection kit for porcine epidemic diarrhea virus, comprising the above-mentioned detection primer set.

[0012] Furthermore, the detection kit includes a first primer set and a second primer set, wherein the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.61 to 120.

[0013] On the other hand, the present invention provides a method for preparing a sequencing fragment of porcine epidemic diarrhea virus, wherein the cDNA obtained by reverse transcription of the sample to be detected is amplified using the above primer set, and the amplified product is purified to obtain a sequencing fragment.

[0014] Further, the cDNA obtained by reverse transcription of the sample to be tested is amplified using the first primer set and the second primer set, and the amplified product is purified to obtain a sequencing fragment;

[0015] The first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 61 to 120.

[0016] Furthermore, the sequencing samples are suitable for second-generation or third-generation sequencing.

[0017] Further, the samples to be tested include clinical samples or environmental samples;

[0018] Preferably, the clinical sample comprises a throat swab, a nasal swab, a blood sample or lung tissue cells.

[0019] On the other hand, the present invention provides the use of the aforementioned primer set, detection reagent or kit in non-disease diagnosis detection of porcine epidemic diarrhea virus.

[0020] Furthermore, the above detection step includes:

[0021] 1) The cDNA obtained by reverse transcription of the sample to be tested is amplified using the above primer set, and the amplified product is purified to obtain a sequencing fragment.

[0022] 2) Using the first primer set and the second primer set to perform multiplex PCR amplification on the cDNA obtained by reverse transcription of the sample to be tested, respectively; wherein the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1~60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.61~120.

[0023] 3) The amplification products of the two sets of primers are combined and purified to obtain sequencing fragments.

[0024] 4) Sequencing to obtain the genome fragment sequence of porcine epidemic diarrhea virus.

[0025] 5) The full-length genome sequence of porcine epidemic diarrhea virus is obtained by alignment and / or assembly with the reference genome.

[0026] Furthermore, the sequencing in step 4) is second-generation or third-generation sequencing.

[0027] Furthermore, the non-disease diagnostic test for porcine epidemic diarrhea virus is to detect environmental samples or throat swabs, nasal swabs, and blood samples of inanimate biological individuals.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] According to the whole genome sequence of PEDV published in the gene sequence database GenBank of the American Center for Biotechnology Information, 60 pairs of amplification primers were designed according to the genome position, and the specific sequences are shown in SEQ ID NO.1~120, and the primer combination of multiple PCR was optimized. The detection method of the present invention can obtain the whole genome sequence information of PEDV of different genotypes. The primer amplification and sequencing results show that the coverage is relatively uniform, and the minimum depth can meet the requirements of accurate detection. The present invention utilizes the advantages of nanopore sequencing in real time, portability, long read length, and high throughput to achieve rapid differential diagnosis of PEDV infection and epidemic; at the same time, its whole genome sequence is obtained, and the obtained genome sequence can provide a scientific basis for virus tracing, pathogen mutation tracking, new strain identification and early warning, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0031] Figure 1 The genome is divided into two groups of amplification products at the 5' end and 3' end according to the primer binding position, and the sequencing depth coverage is shown.

[0032] Figure 2 The sequencing depth coverage of two groups of amplification products formed by random combination of primer pairs.

[0033] Figure 3 The depth coverage of MinION sequencing of G1 PEDV samples.

[0034] Figure 4 The depth coverage of MinION sequencing of G2 PEDV samples.

[0035] Figure 5 This is the sequencing depth coverage of the primer amplification product of Document 1 in Example 3.

[0036] Figure 6 This is the sequencing depth coverage of the primer amplification product of Document 2 in Example 3. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0038] The nucleotide sequences of the primers in the examples are shown in the following table:

[0039]

[0040] Example 1 Primer design for whole genome amplification of PEDV third-generation sequencing

[0041] According to the whole genome sequence of PEDV published in the gene sequence database GenBank of the American Center for Biotechnology Information, molecular biology tools such as BioEdit were used to perform whole genome multiple sequence alignment analysis to obtain its conserved regions, and Primer BLAST was used for primer design and verification. In order to achieve whole genome amplification, multiple pairs of primers were designed for the PEDV genome. The primer information is shown in Table 1. The detection primer set can achieve relatively uniform coverage of PEDV, provide a good technical means for obtaining more uniform and effective PEDV genome sequencing samples, and achieve good results in nanopore sequencing and high-throughput sequencing. The detection primer set is suitable for preparing products for detecting PEDV and is widely used. Specifically, it can be a reagent or a kit. The present invention also protects the reagent or kit, specifically using the above-mentioned detection primer set as primers to amplify and prepare sequencing fragments of the whole genome of PEDV.

[0042] Example 2 Interference with the establishment of a PEDV real-time sequencing method for nanopore sequencing

[0043] This example uses two PEDV strains with different branches and uses the preparation method provided by the present invention to sequence the PEDV samples. The specific process is as follows:

[0044] 1) Nucleic acid extraction: Use Biori automated nucleic acid extraction machine to extract sample nucleic acid. After nucleic acid extraction, Qubit fluorescence quantification instrument is needed to measure the concentration.

[0045] 2) First-strand cDNA synthesis: Take 8μL RNA (concentration 200ng / μL) in a new 0.2mL PCR tube, add 2μL RTMix (TaKaRa) on ice, flick to mix, centrifuge to centrifuge the liquid to the bottom of the tube, without bubbles, and place the PCR tube / plate on ice. Place it in a PCR instrument and run according to the PCR program (25℃2min; 55℃10min; 95℃10min; 4℃Hold), and store at 4℃.

[0046] 3) PCR multiplex amplification: Divide the reaction into two reactions according to tube 1 (first primer set) and tube 2 (second primer set). Use High-Fidelity 2X Master Mix of NEB (NEW ENGLAND BioLabs) to react the same sample under the following conditions: 98°C, 30 s; 35 cycles of: 98°C, 15 s, 65°C, 5 min; and store at 4°C.

[0047] 4) PCR product purification: Combine the products of the two amplification pools into a 1.5 mL EP tube. Use 0.8× volume of magnetic beads for purification, rinse twice with 200 μL of freshly prepared 70% ethanol, and elute the purified product with nuclease-free water.

[0048] 5) PCR product quality inspection: Use Qubit® dsDNA HS Assay Kit to quantify the purified PCR product according to the instructions of the quantification kit.

[0049] 6) Third-generation sequencing: The nanopore sequencer MinION was used to construct a library and sequence the mixed samples of amplified products.

[0050] 7) Analysis of results: Bioinformatics methods were used to align the sequencing fragments with an average depth of 200× to the GenBank reference genome, and the coverage depth of each genomic site was calculated.

[0051] Experimental Example 1

[0052] The genome was divided into 5' and 3' ends for multiplex PCR amplification, specifically

[0053] In step 2), tube 1 of multiplex PCR contains the primer set at the 5' end, SEQ ID NO.1~SEQ ID NO.15, SEQ ID NO.31~SEQ ID NO.45, SEQ ID NO.61~SEQ ID NO.75, SEQ ID NO.91~SEQ ID NO.105, and tube 2 contains the remaining primer set, i.e., the primer set at the 3' end. Only the G1 subtype PEDV sample was sequenced, and the other conditions remained unchanged according to the above experimental steps.

[0054] The results are as follows Figure 1 As shown in the figure, the overall reads coverage is extremely uneven, and some sites have coverage lower than 100 or even 0, which makes it difficult to meet the requirements of full genome coverage.

[0055] Experimental Example 2

[0056] The difference from Experimental Example 1 is that the primer pairs are randomly combined and divided equally into 2 tubes, and only the G2 subtype PEDV samples are sequenced, and the other conditions remain unchanged according to the above experimental steps.

[0057] The results are as follows Figure 2 As shown in the figure, the overall reads coverage is extremely uneven, and some sites have coverage lower than 100 or even 0, which makes it difficult to meet the requirements of full genome coverage.

[0058] Experimental Example 3

[0059] Tube 1 is the first primer set, and tube 2 is the second primer set, which are divided into two reactions. Other conditions are carried out according to the above experimental steps.

[0060] The results showed that the amplification system achieved 100% coverage of the PEDV genome and achieved a coverage depth of >100 times in all amplified regions. The MinION sequencing results were as follows: Figures 1-2 As shown ( Figure 3 This is a G1 subtype PEDV sample. Figure 4 The results show that the standard evaluation of the present invention can meet the needs and provide good quality control for obtaining more uniform and effective PEDV genome sequencing samples.

[0061] Experimental Example 4

[0062] Due to the long sequencing length of MinION, the read length of MinION is usually between 1,000 and 10,000 base pairs. We tried to perform multiplex PCR and third-generation sequencing on the published segmented amplification primers with amplification length of several hundred to several thousand bp (Reference 1: CN106834282A; Reference 2: Cheng Xiaona. Sequence analysis and antigenic epitope expression of epidemic strains of porcine epidemic diarrhea virus (PEDV) [D]. Hainan University, 2015.), and compared them with the present invention. The primers for segmented amplification of the whole genome of PEDV published in the above two documents were divided into two groups for multiplex PCR. The PCR products of each group did not overlap. Finally, the two groups of PCR products were mixed and sequenced, and only the G1 subtype PEDV samples were sequenced. Results ( Figure 5 , 6) showed that under multiplex PCR conditions, the reported segmented amplification primer sets, whether long or short amplification products, had coverage of some sites below 100, or even 0, and the overall reads coverage was extremely uneven, making it difficult to meet the requirements for full genome coverage.

[0063] The above results show that multiplex PCR combined with nanopore sequencing still requires the design of primers of appropriate length and appropriate sites, and the combination of primer pairs that need to be verified can achieve the best effect.

[0064] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A detection primer set for porcine epidemic diarrhea virus, characterized in that: The detection primer set includes 60 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 120.

2. Use of the detection primer set according to claim 1 in the preparation of a product for detecting porcine epidemic diarrhea virus.

3. The use according to claim 2, characterized in that: The product comprises a reagent or a kit.

4. A porcine epidemic diarrhea virus detection reagent, characterized in that: Comprising the detection primer set described in claim 1.

5. The detection reagent according to claim 4, characterized in that The detection reagent includes a first primer set and a second primer set, wherein the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.61 to 120.

6. A detection kit for porcine epidemic diarrhea virus, characterized in that: Comprising the detection primer set described in claim 1.

7. The detection kit according to claim 6, characterized in that The detection kit includes a first primer set and a second primer set, wherein the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.61 to 120.

8. A method for preparing a sequencing fragment of porcine epidemic diarrhea virus, characterized in that: The cDNA obtained by reverse transcription of the sample to be detected is amplified using the detection primer set described in claim 1, and the amplified product is purified to obtain a sequencing fragment.

9. The preparation method according to claim 8, characterized in that: The cDNA obtained by reverse transcription of the sample to be tested is amplified using the first primer set and the second primer set, and the amplified products are purified to obtain sequencing fragments; wherein, the first primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1~60; the second primer set includes 30 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.61~120.

10. Use of the primer set of claim 1, the detection reagent of claim 3 or the kit of claim 6 in non-disease diagnosis detection of porcine epidemic diarrhea virus.

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