Porcine delta coronavirus whole genome sequencing method based on nanopore sequencing

Abstract

The invention provides an amplification primer and method for a porcine delta coronavirus whole genome. The novel coronavirus detection primer group comprises 124 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-248. According to the detection primer group provided by the invention, relatively uniform coverage on the porcine deltacoronavirus 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 deltacoronavirus 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 shortened, infection of the porcine deltacoronavirus can be rapidly identified and diagnosed, the whole genome sequence of the porcine deltacoronavirus 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 application relates to the field of animal virology, and provides a porcine deltacoronavirus whole genome amplification primer and method. BACKGROUND

[0002] Porcine deltacoronavirus (PDCoV) belongs to the newly identified delta coronavirus genus in the Coronaviridae family, which was first discovered by Hong Kong scholars from pig fecal samples in 2012, but at that time, in-depth research on virus isolation and pathogenicity was not carried out. In February 2014, American scholars detected PDCoV from a pig farm with diarrhea and successfully isolated the virus. Animal experiments confirmed that the isolated strain had strong pathogenicity to newborn piglets, and infected piglets showed decreased appetite, vomiting, severe diarrhea, etc. Subsequently, at least 20 states in the United States reported the prevalence of PDCoV. After the United States reported PDCoV, Canada, South Korea, Thailand, Vietnam, Laos and other countries also detected the prevalence of PDCoV from pig diarrhea samples, and reports showed that the Thai strain had strong pathogenicity to piglets, growing pigs and sows, causing the death of a large number of piglets, which attracted worldwide attention of the pig industry. So far, more than 25 provinces in China have reported PDCoV diarrhea outbreaks, indicating that the virus exists widely and spreads rapidly in pig farms across the country.

[0003] PDCoV can infect pigs of all ages and cause disease in production, and has strong pathogenicity to piglets, leading to the death of a large number of piglets. This disease occurs in all seasons, mostly in winter and spring. PDCoV continuously evolves and recombines in the field, and the virus can be transmitted across species through intermediate hosts, which is a potential zoonotic infectious disease. Rapid identification and diagnosis of PDCoV infection and obtaining its genomic information can provide a basis for early detection, early control and tracing of the virus, and play a crucial role in preventing the outbreak of PDCoV epidemic. At present, the virus detection methods mainly include virus isolation and culture, immunological detection and nucleic acid detection, but the above three methods cannot obtain the complete genome sequence of the virus and cannot analyze the characteristics of the viral genome. Nanopore sequencing has the characteristics of real-time, portable, long read, high throughput, etc., and can realize real-time rapid detection on site; however, there is no PDCoV sequencing technology designed based on the advantages of nanopore sequencing. Therefore, there is still an urgent need for a porcine deltacoronavirus whole genome sequencing technology based on third-generation nanopore sequencing. SUMMARY

[0004] The application designs PDCoV whole genome long PCR amplification specific primers, and further provides a nanopore sequencing PDCoV real-time detection method, which fully gives play to the advantages of nanopore sequencing in pathogen detection, such as real-time, portable, long read, high throughput and the like. The detection method can quickly diagnose and differentiate PDCoV infection, and at the same time obtain the whole genome sequence thereof, and the obtained whole genome sequence can provide information for pathogen variation monitoring and molecular epidemiology research.

[0005] To achieve the above-mentioned purposes, the technical scheme adopted by the application is:

[0006] A detection primer set of a novel coronavirus, the detection primer set comprising 124 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 1-248.

[0007] The application of the above-mentioned detection primer set in the preparation of a product for detecting a novel coronavirus.

[0008] Further, the product comprises a reagent or a kit.

[0009] On the other hand, the application provides a detection reagent for a novel coronavirus, comprising the above-mentioned detection primer set.

[0010] Further, the detection reagent comprises a first primer set and a second primer set, wherein the first primer set comprises 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 1-124; the second primer set comprises 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 125-248.

[0011] On the other hand, the application provides a detection kit for a novel coronavirus, comprising the above-mentioned detection primer set.

[0012] Further, the detection kit comprises a first primer set and a second primer set, wherein the first primer set comprises 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 1-124; the second primer set comprises 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 125-248.

[0013] On the other hand, the application provides a preparation method of a sequencing fragment of a novel coronavirus, which uses the above-mentioned detection primer set to amplify the cDNA obtained by reverse transcription of a to-be-detected sample, and purifies the amplification product to obtain the sequencing fragment.

[0014] Further, the first primer set and the second primer set are used to amplify the cDNA obtained by reverse transcription of a to-be-detected sample, respectively, and the amplification product is purified to obtain the sequencing fragment.

[0015] The first primer set includes 62 pairs of amplification primers, and specific sequences are shown in SEQ ID NO. 1-124; and the second primer set includes 62 pairs of amplification primers, and specific sequences are shown in SEQ ID NO. 125-248.

[0016] Further, the sequencing sample is suitable for second-generation or third-generation sequencing.

[0017] Further, the sample to be detected includes a clinical sample or an environmental sample.

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

[0019] In another aspect, the present application provides an application of the aforementioned primer set, detection reagent or kit in non-disease diagnosis detection of porcine deltacoronavirus.

[0020] Further, the detection step includes:

[0021] 1) extracting total DNA of the sample.

[0022] 2) performing multiplex PCR amplification on the DNA obtained from the sample to be detected by using the first primer set and the second primer set respectively, and obtaining sequencing fragments by purifying the amplification products; wherein the first primer set includes 62 pairs of amplification primers, and specific sequences are shown in SEQ ID NO. 1-124; and the second primer set includes 62 pairs of amplification primers, and specific sequences are shown in SEQ ID NO. 125-248.

[0023] 3) combining the amplification products of the two primer sets, and obtaining sequencing fragments by purification.

[0024] 4) obtaining the sequence of the genomic fragment by sequencing.

[0025] 5) obtaining the genomic sequence of porcine deltacoronavirus by comparison with a reference genome and / or assembly.

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

[0027] Further, the non-disease diagnosis detection of porcine deltacoronavirus is detection of a throat swab, a nasal swab, a blood sample or lung tissue cells of an environmental sample or an inanimate biological individual.

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

[0029] According to the published whole genome sequence of PDCoV in GenBank of the National Center for Biotechnology Information, 124 pairs of amplification primers are designed according to the genomic position, and the specific sequences are shown as SEQ ID NO. 1-248. The detection method of the present application can obtain the whole genome sequence information of PDCoV. The present application utilizes the advantages of real-time, portable, long read length and high throughput of nanopore sequencing to realize rapid differential diagnosis of PDCoV infection; at the same time, the whole genome sequence is obtained, and the obtained genome sequence can provide scientific basis for virus tracing, pathogen variation tracking, new strain identification and early warning, etc. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 For the sequencing depth coverage of the two groups of amplification products according to the primer binding position, the genome is divided into 5' end and 3' end.

[0031] Figure 2 For the sequencing depth coverage of the two groups of amplification products randomly combined by primer pairs.

[0032] Figure 3 For the MinION sequencing depth coverage of PDCoV strain branch 1 sample.

[0033] Figure 4 For the MinION sequencing depth coverage of PDCoV strain branch 2 sample.

[0034] The present application will be further explained in conjunction with specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not intended to limit the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0037] 1. PDCoV three-generation sequencing whole genome amplification primer design

[0038] According to the whole genome sequence of PDCoV disclosed in the GenBank of the United States Biotechnology Information Center gene sequence database, the whole genome multiple sequence alignment analysis is carried out by using BioEdit and other molecular biology tools to obtain the conserved region, and the primer design and verification are carried out by using Primer-BLAST. A plurality of primers are designed for PDCoV genome to realize whole genome amplification. The primer information is as follows:

[0039] Table 1 Sequence of the detection primer set involved in the application

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] The detection primer set can realize the relatively uniform coverage of PDCoV, and provides a good technical means for obtaining more uniform and effective PDCoV genome sequencing samples, and good results are obtained in nanopore sequencing and high-throughput sequencing. The detection primer set is suitable for being prepared into a product for detecting PDCoV and is widely used. Specifically, it can be a reagent or a kit. The application also protects the reagent or the kit, specifically, the above-mentioned detection primer set is used as a primer to amplify and prepare the sequencing fragment of the whole genome of PDCoV.

[0047] 2. Establishing a real-time sequencing method of PDCoV for nanopore sequencing

[0048] In this embodiment, two PDCoV strains with different branches are used to sequence the PDCoV sample by using the preparation method provided by the application, and the specific process is as follows:

[0049] 1) Synthesis of single-strand cDNA: take 8 μL of RNA in a new 0.2 mL PCR tube, add 2 μL of RTMix (TaKaRa) on ice, mix gently, centrifuge the liquid to the bottom of the tube without bubbles, and place the PCR tube / plate on ice. Place on the PCR instrument according to the PCR program (25℃ 2min; 55℃ 10min; 95℃ 10min; 4℃ Hold) operation, 4℃ storage.

[0050] 2) PCR multiplex amplification: two reactions according to tube 1 (first primer set) and tube 2 (second primer set) were performed using NEB (NEW ENGLAND BioLabs) High-Fidelity 2X Master Mix, conditions: 98℃, 30s; 35 cycles: 98℃, 15s, 65℃, 5min; 4℃ storage.

[0051] 3) PCR product purification: the products of the two amplification pools were combined into a 1.5ml EP tube. 0.8x volume of magnetic beads were used for purification, 200ul freshly prepared 70% ethanol was used for rinsing twice, and then the purified product was eluted with nuclease-free water.

[0052] 4) PCR product quantification: dsDNA HS Assay Kit was used to quantify the PCR purified product according to the operation instructions of the quantitative kit.

[0053] 5) Third-generation sequencing: the MinION nanopore sequencer was used to construct a library and sequence the mixed sample of the amplification product.

[0054] 6) Analysis results: bioinformatics methods were used to align the sequence of the sequencing fragment to the GenBank reference genome, and calculate the coverage depth of each genomic site.

[0055] Experimental Example 1

[0056] The genome was divided into 5' and 3' ends for multiplex PCR amplification, specifically according to the binding sites of the PCR primers on the sequence (allowing 3 mismatches, 3' end not allowing mismatches) the primers were divided into genomic 5' end primer set (PCR tube 1, expected to amplify 5' end to about 12700bp) and genomic 3' end primer set (PCR tube 2, expected to amplify 3' end from about 12600bp). Only PDCoV strain branch 1 sample sequencing, other conditions unchanged according to the above experimental steps.

[0057] The results are shown in Figure 1 Overall reads coverage is extremely uneven, and some sites have coverage less than 100, even 0, which is difficult to meet the requirements of whole genome coverage.

[0058] Experimental Example 2

[0059] The difference from Experimental Example 1 is that the primers are randomly combined into two tubes, only PDCoV strain branch 2 sample sequencing, other conditions unchanged according to the above experimental steps.

[0060] The results are shown in Figure 2 ​As shown, the whole reads cover extremely unevenly, and some sites have coverage less than 100, even 0, which is difficult to meet the requirement of whole genome coverage.

[0061] Experimental Example 3

[0062] According to Table 1, the first primer group and the second primer group are grouped, two reactions are divided, and other conditions are performed according to the above experimental procedures.

[0063] The results show that the amplification system realizes 100% coverage of the PDCoV genome, and all the amplification regions achieve a coverage depth of more than 100 times. The MinION sequencing results are as shown in Figures 3-4 Figure 3 PDCoV strain branch 1 samples, Figure 4 PDCoV strain branch 2 samples). The results show that the standard evaluation of the present application can meet the requirements and provide good quality control for obtaining more uniform and effective PDCoV genome sequencing samples.

[0064] The above results show that multiplex PCR combined with nanopore sequencing still needs to design primers with appropriate length and appropriate site, and the primer pair combination needs to be verified to achieve the best effect.

[0065] The above description of the disclosed embodiments enables one skilled in the art to make or use the application. Various modifications to the above-described embodiments will be apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application will not be limited to the embodiments shown herein but will be accorded the widest scope consistent with the principles and novel features disclosed herein.​

Claims

1. A detection primer set for porcine delta coronavirus, characterized in that: The detection primer set includes 124 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 1-248.

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

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

4. A detection reagent for porcine delta coronavirus, characterized in that The invention comprises the detection primer set according to 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 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1-124; the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 125-248.

6. A detection kit for porcine delta coronavirus, characterized in that: The invention comprises the detection primer set according to 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 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1-124; the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 125-248.

8. A method for preparing a porcine delta coronavirus sequencing fragment, characterized in that: The detection primer set according to claim 1 is used to amplify the cDNA obtained by reverse transcription of 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; Among them, the first primer set includes 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1-124; the second primer set includes 62 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.125-248.

10. Use of the primer set of claim 1, the detection reagent of claim 3, or the kit of claim 6 in non-disease diagnostic detection of porcine deltacoronavirus.

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