Chicken Marek's virus whole genome sequencing method based on nanopore sequencing

Abstract

The invention provides an amplification primer and method for a chicken Marek's virus whole genome. The detection primer group for the chicken Marek's virus comprises 206 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-412. The detection primer group provided by the invention can realize relatively uniform coverage of the chicken Marek's virus, and 100% region coverage of a genome is realized in nanopore sequencing. The sequencing fragment preparation method of the chicken Marek's 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 chicken Marek's virus can be identified and diagnosed at the soonest 15 h, meanwhile, the whole genome sequence of the chicken Marek's virus is obtained, and the method is suitable for 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 present invention relates to the field of animal virology and provides primers and a method for amplifying the whole genome of chicken Marek's virus. Background Art

[0002] Marek's disease (MD) in chickens is a highly contagious disease caused by the Marek's disease virus (MDV). Affected chickens display clinical symptoms such as lethargy, paralysis, ataxia, wing drooping, freezing, and ruffled fur. Autopsy reveals lymphoproliferative tumors in viscera and other organs. MD can also cause immunosuppression in affected birds, leading to secondary infection with other pathogens. MD is a worldwide epidemic, resulting in direct economic losses of US$1 to 2 billion annually. Vaccination is the primary means of controlling the disease. However, MD vaccines are non-clearing and cannot prevent infection and excretion of wild-type strains. This leads to the long-term coexistence of wild-type and vaccine strains in the same organism, promoting the evolution of virulence in the wild-type strain. Over the past few decades, MDV has continued to evolve, and the protective effects of the MD vaccines used in the past have been suboptimal.

[0003] Therefore, it is necessary to understand MDV infection and prevalence in chickens and analyze the pathogenic characteristics and virulence variation of wild-type MDV strains in my country. Rapid differential diagnosis of MDV infection and obtaining genomic information can provide a basis for early detection, control, and source tracing of the virus, thereby minimizing the economic losses to the poultry industry. Nanopore sequencing offers the advantages of real-time, portability, long read length, and high throughput, enabling rapid, on-site detection. However, nanopore sequencing has limitations. For example, compared with first-generation, second-generation, and third-generation PacBio sequencing, nanopore sequencing has lower accuracy, particularly when the library fragments are too long, resulting in a significant decline in sequencing quality. Genome segmentation amplification may address these issues. However, currently, no MDV sequencing technology designed for nanopore sequencing exists. Therefore, there is an urgent need for a third-generation nanopore-based MDV whole-genome sequencing technology that can rapidly obtain the pathogen's complete genome sequence. This is of vital importance for both basic research on MDV and the prevention and control of MDV. Summary of the Invention

[0004] This study designed specific primers for PCR amplification of the entire MDV genome and further provides a real-time MDV detection method using nanopore sequencing, leveraging the advantages of nanopore sequencing for pathogen detection, including real-time, portability, long read length, and high throughput. This detection method can rapidly identify and diagnose MDV infection and simultaneously obtain the full genome sequence (including all coding genes and their intergenic regions). The obtained full genome sequence can provide information for pathogen variation monitoring and molecular epidemiological studies.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A detection primer set for chicken Marek's virus comprises 206 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 412.

[0007] Application of the above detection primer set in the preparation of products for detecting chicken Marek's virus.

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

[0009] In another aspect, the present invention provides a detection reagent for chicken Marek's 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 104 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.209 to SEQ ID NO.412.

[0011] In another aspect, the present invention provides a detection kit for chicken Marek's 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 104 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.209 to SEQ ID NO.412.

[0013] On the other hand, the present invention provides a method for preparing a sequencing fragment of chicken Marek's 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] Furthermore, the DNA obtained from the sample to be tested is subjected to multiplex PCR amplification using the first primer set and the second primer set, and the amplified products are purified to obtain sequencing fragments;

[0015] Among them, the first primer set includes 104 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.209 to SEQ ID NO.412.

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

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

[0018] Preferably, the clinical sample includes feather marrow or anticoagulated blood.

[0019] In another aspect, the present invention provides use of the aforementioned primer set, detection reagent or kit in non-disease diagnostic detection of chicken Marek's virus.

[0020] Furthermore, the above detection step includes:

[0021] 1) Extract total DNA from samples.

[0022] 2) Multiplex PCR amplification was performed on the DNA obtained from the sample to be tested using the first primer set and the second primer set, and the amplified products were purified to obtain sequencing fragments; wherein the first primer set includes 104 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO. 1 to SEQ ID NO. 208; and the second primer set includes 102 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO. 209 to SEQ ID NO. 412.

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

[0024] 4) Sequencing to obtain the genome fragment sequence.

[0025] 5) Obtaining the genome sequence of chicken Marek's virus by alignment and / or assembly with a reference genome.

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

[0027] Furthermore, the non-disease diagnostic test for chicken Marek's virus is to detect environmental samples or feather marrow or anticoagulated blood of inanimate biological individuals.

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

[0029] Based on the published full genome sequence of MDV in GenBank, the gene sequence database of the Center for Biotechnology Information (CBI), 206 pairs of multiplex PCR amplification primers were designed according to genomic location. The specific sequences are shown in SEQ ID NOs. 1-412. The detection method of the present invention can obtain full genome sequence information of MDV. Leveraging the real-time, portable, long-read, and high-throughput advantages of nanopore sequencing, the present invention enables rapid differential diagnosis of MDV infection in as little as 15 hours. Simultaneously, the full genome sequence is obtained, providing a scientific basis for tracing the virus's origin, tracking pathogen mutations, identifying new strains, and providing early warning. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 2 The sequencing depth coverage of two groups of amplification products obtained by randomly combining primer pairs.

[0032] Figure 3 To implement the depth coverage of MinION sequencing of 814 strain samples.

[0033] Figure 4 To implement the depth coverage of MinION sequencing of CVI988 strain samples.

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

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0037] 1. Design of primers for whole genome amplification of MDV third-generation sequencing

[0038] Based on the published MDV genome sequence in GenBank, the Center for Biotechnology Information's gene sequence database, molecular biology tools such as BioEdit were used to perform whole-genome multiple sequence alignment analysis to identify conserved regions. Primer3 was then used for primer design and verification. Multiple primer pairs were designed to achieve whole-genome amplification of the MDV genome. Primer information is as follows:

[0039] Table 1 Detection primer set sequences involved in the present invention

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] This detection primer set can achieve relatively uniform coverage of MDV, providing an excellent technical means for obtaining more uniform and effective MDV genome sequencing samples. It has achieved good results in both nanopore sequencing and high-throughput sequencing. This detection primer set is suitable for preparation into products for detecting MDV and is widely applicable. Specifically, it can be a reagent or kit. The present invention also protects such a reagent or kit, which specifically uses the above-mentioned detection primer set as primers to amplify and prepare sequencing fragments of the entire MDV genome.

[0047] 2. Establishment of a real-time sequencing method for MDV using nanopore sequencing

[0048] This example uses the preparation method provided by the present invention to sequence MDV samples. The specific process is as follows:

[0049] 1) Nucleic acid extraction: Use Biori automated nucleic acid extraction machine to extract sample nucleic acid. After nucleic acid extraction, the concentration needs to be measured using Qubit fluorescence quantification instrument.

[0050] 2) Multiplex PCR Amplification: Two reactions were performed in tube 1 (first primer set) and tube 2 (second primer set). The same sample was subjected to two reactions using NEB (NEB) High-Fidelity 2X Master Mix under the following conditions: 98°C for 30 s, 35 cycles of 98°C for 15 s, 65°C for 5 min, and storage at 4°C.

[0051] 3) PCR product purification: Combine the products of the two amplification pools into a 1.5 ml EP tube. Purify using 0.8x volume of magnetic beads, rinse twice with 200 μl of freshly prepared 70% ethanol, and elute the purified product with nuclease-free water.

[0052] 4) PCR product quality inspection: use HS Assay Kit double-stranded DNA fluorescence quantification kit was used to quantify the PCR purified product according to the operating instructions of the quantification kit.

[0053] 5) Third-generation sequencing: The nanopore sequencer MinION was used to construct and sequence the mixed samples of amplified products, with an average sequencing data volume of no less than 500×.

[0054] 6) Analysis of results: Using bioinformatics methods, the sequence of the sequencing fragments was aligned to the GenBank reference genome, and the coverage depth of each genomic site was calculated.

[0055] Experimental Example 1

[0056] Multiplex PCR amplification was performed separately for the 5' and 3' ends of the genome. Specifically, the primers were divided into a 5' primer set (PCR tube 1) and a 3' primer set (PCR tube 2) based on their binding sites along the sequence (three mismatches were allowed, while no mismatches were allowed at the 3' end). For strain 814 (JF742597), the 5' primer set was expected to amplify to 88,043 bp, while the 3' primer set was expected to amplify from 87,933 bp. There was some overlap between the amplified regions of the two primer sets. All other conditions remained the same as in the previous experimental steps.

[0057] 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.

[0058] Experimental Example 2

[0059] The difference from Experimental Example 1 is that the primer pairs were randomly combined and divided equally into two tubes, and only the CVI988 strain (PV035744) sample was sequenced. Other conditions remained unchanged according to the above experimental steps.

[0060] 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.

[0061] Experimental Example 3

[0062] According to Table 1, the primers were grouped into the first primer set and the second primer set, and the reactions were divided into two. Other conditions were carried out according to the above experimental steps.

[0063] The results showed that the amplification system achieved 100% coverage of the MDV genome and achieved a coverage depth of >100 times in all amplified regions. The MinION sequencing results were as follows: Figures 3-4 As shown ( Figure 3 This is a sample of strain 814. Figure 4 This result shows that the standard evaluation of the present invention can meet the needs and provide good quality control for obtaining more uniform and effective MDV genome sequencing samples.

[0064] The above results indicate 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 needs to be verified to achieve the best effect.

[0065] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for detecting chicken Marek's virus, characterized in that: The detection primer set includes 206 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO. 1-412.

2. Use of the detection primer set according to claim 1 in preparing a product for detecting chicken Marek's virus.

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

4. A detection reagent for chicken Marek's virus, characterized in that: Comprising 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 104 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.209 to SEQ ID NO.

412.

6. A detection kit for chicken Marek's virus, characterized in that: Comprising 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 104 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.209 to SEQ ID NO.

412.

8. A method for preparing a chicken Marek's virus sequencing fragment, 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 first primer set and the second primer set are used to amplify the DNA nucleic acid extracted from the sample to be tested, and the amplified products are purified to obtain sequencing fragments; Among them, the first primer set includes 104 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1 to SEQ ID NO.208; the second primer set includes 102 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.209 to SEQ ID NO.

412.

10. Use of the primer set according to claim 1, the detection reagent according to claim 3, or the kit according to claim 6 in non-disease diagnostic detection of chicken Marek's virus.

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