Porcine pseudorabies virus whole genome sequencing method based on nanopore sequencing

Abstract

The invention provides an amplification primer and method for a porcine pseudorabies virus whole genome. The detection primer group for the porcine pseudorabies virus comprises 271 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-542. According to the detection primer group provided by the invention, relatively uniform coverage on the porcine pseudorabies virus can be realized, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the porcine pseudorabies virus whole genome is simple, convenient and easy to operate, the amplification effect is good, nanopore sequencing has the advantage of simultaneous sequencing and real-time analysis, the detection time can be greatly shortened, porcine pseudorabies virus infection can be identified and diagnosed at the soonest 15 hours, meanwhile, the whole genome sequence of the porcine pseudorabies virus is obtained, and the method is suitable for large-scale popularization and application. 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 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 pseudorabies virus. Background Art

[0002] Pseudorabies (PR) is an acute contact infectious disease caused by pseudorabies virus (PRV) in porcine animals. The main symptoms are neurological symptoms and death in newborn piglets after infection. Pregnant mothers may suffer from miscarriage, stillbirth, mummification and respiratory symptoms. Fattening pigs mainly show respiratory disorders and slow growth. Since the virus was discovered by Hungarian scholar Aueszky in 1902, it has caused huge economic losses to the pig industry worldwide. So far, the virus has appeared for more than 100 years, but it has not been effectively eradicated in many pig-raising countries. In 1947, Mr. Liu Yongchun, a pioneer in my country's veterinary industry, first discovered a case of pseudorabies virus infection in cats, confirming the existence of the disease in my country. In the decades since then, PRV has caused huge losses to my country's pig industry, especially since 2011, the emergence and widespread spread of variant pseudorabies viruses have seriously restricted the healthy development of the pig industry.

[0003] In the 1990s, my country introduced the classic attenuated vaccine Bartha-K61 strain of PRV from Hungary. The vaccine has weak toxicity and good immune effect. After mass production and wide application in major pig farms, the prevalence of pseudorabies in my country has been effectively controlled. Referring to the purification method of PRV in the United States, many pig farms have also completed the purification of the pathogen. However, in 2011, the domestic PRV epidemic strain mutated again. The newly generated strain and the classic PRV attenuated vaccine Bartha-K61 belong to different subgroups, making the existing vaccine unable to produce sufficient protection, which led to the re-epidemic of PRV in China. In recent years, PRV has been in a state of mutation and evolution. Rapid differential diagnosis of PRV 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. Nanopore sequencing has the characteristics of real-time, portability, long read length, and high throughput. It can realize real-time rapid detection on site, timely and accurately identify pathogens, and give accurate detection results; however, there is currently no sequencing technology for PRV designed for the advantages of nanopore sequencing. Therefore, there is still an urgent need for a PRV whole genome sequencing technology based on third-generation nanopore sequencing to quickly obtain the whole genome sequence of pathogens, providing an important reference and theoretical basis for viral genetic variation and epidemiological analysis. Summary of the invention

[0004] The present invention designs specific primers for PCR amplification of the whole genome of PRV, and further provides a real-time detection method for PRV 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 identify and diagnose PRV infection, and simultaneously obtain its whole genome sequence, which 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 pseudorabies virus of porcine, the detection primer set comprising 271 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 542.

[0007] In another aspect, the present invention provides use of the above detection primer set in preparing a product for detecting porcine pseudorabies virus.

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

[0009] In another aspect, the present invention provides a detection reagent for porcine pseudorabies 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 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

[0011] In another aspect, the present invention provides a detection kit for porcine pseudorabies 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 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

[0013] On the other hand, the present invention provides a method for preparing a sequencing fragment of porcine pseudorabies virus, wherein the DNA extracted from the sample to be detected is amplified using the above-mentioned detection primer set, and the amplified product is purified to obtain a sequencing fragment.

[0014] Further, the DNA obtained from 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] Among them, the first primer set includes 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

[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 includes blood, semen, tonsil, etc.

[0019] Furthermore, the above detection step includes:

[0020] 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.

[0021] 2) Using the first primer set and the second primer set, respectively, multiplex PCR amplification is performed on the cDNA obtained by reverse transcription of the sample to be tested; wherein the first primer set includes 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

[0022] 3) The amplification products of the two sets of primers are combined and purified to obtain sequencing fragments.

[0023] 4) Sequencing to obtain the genome fragment sequence of porcine pseudorabies virus.

[0024] 5) Obtain the full-length genome sequence of porcine pseudorabies virus by alignment and / or assembly with the reference genome.

[0025] Furthermore, the non-disease diagnostic test for porcine pseudorabies virus is to detect environmental samples or blood, semen or tonsil samples of inanimate biological individuals.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] According to the whole genome sequence of PRV disclosed in the gene sequence database GenBank of the Center for Biotechnology Information of the United States, 271 pairs of amplification primers were designed according to the genome position, and the specific sequences are shown in SEQ ID NO.1 to 542. The detection method of the present invention can obtain the whole genome sequence information of PRV. The present invention utilizes the advantages of real-time, portable, long read length and high throughput of nanopore sequencing to achieve rapid differential diagnosis of PRV infection, as fast as 15 hours; 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

[0028] 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.

[0029] Figure 1 This is the depth coverage of MinION sequencing of genotype I PRV strain using the comparative primer set in Experimental Example 1 of the present invention.

[0030] Figure 2 This is the depth coverage of MinION sequencing of the genotype II classical PRV strain using the comparative primer set in Experimental Example 2 of the present invention.

[0031] Figure 3 This is the depth coverage of MinION sequencing of genotype II variant PRV strains using the comparative primer set in Experimental Example 3 of the present invention.

[0032] Figure 4 This is the depth coverage of MinION sequencing of genotype I PRV strain using the primer set of the present invention in Experimental Example 4 of the present invention.

[0033] Figure 5 This is the depth coverage of MinION sequencing of the genotype II classical PRV strain using the primer set of the present invention in Experimental Example 4 of the present invention.

[0034] Figure 6 This is the depth coverage of MinION sequencing of genotype II variant PRV strains using the primer set of the present invention in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] The nucleotide sequences of the primers in the examples are shown in the following table:

[0037] Table 1 Detection primer set sequences involved in the present invention

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[0047] Example 1 Primer design for whole genome amplification of PRV third-generation sequencing

[0048] According to the whole genome sequence of PRV disclosed in the gene sequence database GenBank of the Center for Biotechnology Information of the United States, 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 PRV genome. The primer information is shown in Table 1. The detection primer set can achieve relatively uniform coverage of PRV, provide a good technical means for obtaining more uniform and effective PRV 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 PRV 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 PRV.

[0049] Example 2 Establishment of a PRV real-time sequencing method using nanopore sequencing

[0050] This example uses three PRV strains with different branches and uses the preparation method provided by the present invention to sequence the PRV samples. The specific process is as follows:

[0051] 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.

[0052] 2) Synthesis of single-strand cDNA: Take 8 μL RNA (concentration 200 ng / μL) in a new 0.2 mL PCR tube, add 2 μL 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 on a PCR instrument and run it according to the PCR program (25℃2min; 55℃10min; 95℃10min; 4℃Hold), and store it at 4℃.

[0053] 3) PCR multiplex amplification: Tube 1 (first primer set) and tube 2 (second primer set) were divided into two reactions. The same sample was reacted using High-Fidelity 2X Master Mix from NEB (NEW ENGLAND BioLabs) under the following conditions: 98°C, 30 s; 35 cycles: 98°C, 15 s, 65°C, 5 min; and stored at 4°C.

[0054] 4) PCR product purification: The products of the two amplification pools were combined into a 1.5 mL EP tube. 0.8× volume of magnetic beads were used for purification, and the purified products were eluted with nuclease-free water after rinsing twice with 200 μL of freshly prepared 70% ethanol.

[0055] 5) PCR product quality inspection: use dsDNA HS Assay Kit: quantify the purified PCR product according to the instructions of the quantification kit.

[0056] 6) Third-generation sequencing: The nanopore sequencer MinION was used to construct a library and sequence the mixed samples of amplified products.

[0057] 7) Analysis results: Bioinformatics methods were used to align the sequencing fragment sequences to the GenBank reference genome, and the coverage depth of each genomic site was calculated.

[0058] Experimental Example 1

[0059] The genome was divided into 5' and 3' ends for multiplex PCR amplification, specifically

[0060] In step 2), tube 1 of multiplex PCR is the primer set at the 5' end, SEQ ID NO.1 to SEQ ID NO.68, SEQ ID NO.137 to SEQ ID NO.204, SEQ ID NO.273 to SEQ ID NO.340, SEQ ID NO.408 to SEQ ID NO.475, and tube 2 is the remaining primer set, i.e., the primer set at the 3' end. Only genotype I PRV samples were sequenced, and other conditions remained unchanged according to the above experimental steps.

[0061] 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.

[0062] Experimental Example 2

[0063] The difference from Experimental Example 1 is that the primer pairs are randomly combined and divided equally into 2 tubes, and only the genotype II classical PRV samples are sequenced, and other conditions remain unchanged according to the above experimental steps.

[0064] 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.

[0065] Experimental Example 3

[0066] The primer pairs were randomly combined and divided equally into 2 tubes. Only the genotype II variant PRV samples were sequenced, and other conditions remained unchanged according to the above experimental steps.

[0067] The results are as follows Figure 3 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.

[0068] Experimental Example 4

[0069] 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. The MinION sequencing results are as follows Figures 4 to 6 As shown, Figure 4 For genotype I PRV samples, Figure 5 It is a classic PRV sample of genotype II. Figure 6 These are the sequencing results of the PRV sample with genotype II variant.

[0070] The results showed that the amplification system achieved 100% coverage of the PRV genome and achieved a coverage depth of >100 times in all amplified regions. This result shows that the standard evaluation of the present technical invention can meet the needs and provide good quality control for obtaining more uniform and effective PRV genome sequencing samples.

[0071] 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 pseudorabies virus in porcine, characterized in that: The detection primer set includes 271 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 542.

2. Use of the detection primer set according to claim 1 in preparing a product for detecting porcine pseudorabies virus.

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

4. A detection reagent for porcine pseudorabies virus, 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 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

6. A detection kit for porcine pseudorabies 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 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.273 to 542.

8. A method for preparing a sequencing fragment of porcine pseudorabies virus, characterized in that: The detection primer set according to claim 1 is used to amplify the DNA nucleic acid extracted from the sample to be detected, 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 136 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1~272; the second primer set includes 135 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.273~542.

10. The preparation method according to claim 8, characterized in that: The sequencing samples are suitable for third-generation sequencing.

11. The preparation method according to claim 8, characterized in that: The samples to be detected include clinical samples or environmental samples.

12. The preparation method according to claim 11, characterized in that: The clinical samples include blood, semen or tonsils, etc.

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