A method for whole genome sequencing of chicken marek's disease virus based on nanopore sequencing
By designing long PCR amplification primers for the whole genome of MDV and using nanopore sequencing methods, the accuracy problem of nanopore sequencing technology in the detection of Marek's virus in chickens was solved, realizing rapid and accurate whole genome sequencing and detection of MDV, supporting virus tracing and mutation monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WENS FOODSTUFF GROUP CO LTD
- Filing Date
- 2025-06-19
- Publication Date
- 2026-07-21
AI Technical Summary
Existing nanopore sequencing technology has low accuracy in detecting chicken Marek's virus, especially when the library fragment is too long, the sequencing quality drops significantly. There is a lack of whole genome sequencing technology for MDV, which makes it difficult to meet the needs of rapid identification and diagnosis and obtaining viral genome information.
We designed specific primers for long PCR amplification of the entire MDV genome and combined them with nanopore sequencing to obtain sequencing fragments through multiplex PCR amplification and purification, enabling rapid detection and sequence acquisition of the entire MDV genome.
It enables rapid identification and diagnosis of MDV infection, obtains the whole genome sequence, provides information for pathogen variation monitoring and molecular epidemiological research, and has the advantages of real-time, portability, long read length and high throughput, with a detection time of up to 15 hours.
Smart Images
Figure CN120683312B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal virology, and provides primers and methods for amplifying the whole genome of chicken Marek's virus. Background Technology
[0002] Marek's disease (MD) in chickens is a highly contagious disease caused by the Gallid alphaherpesvirus (MDV). Affected chickens exhibit clinical symptoms such as lethargy, paralysis, ataxia, drooping wings, standing still, and ruffled feathers. Necropsy reveals lymphoproliferative tumors in the internal organs and other parts of the body. The disease can also cause immunosuppression in affected chickens, leading to secondary infections with other pathogens. MD is prevalent worldwide, causing direct economic losses of $1-2 billion annually. Vaccination is the primary means of controlling the disease. However, MD vaccination is a non-eliminatory immunization method and cannot prevent infection and shedding of wild-type virus strains. This results in the long-term coexistence of wild-type and vaccine strains in the same organism, promoting the evolution of the wild-type strain's virulence. Over the past few decades, MDV has continuously evolved, and the protective effect of concurrently used MD vaccines has been less than ideal.
[0003] Therefore, it is necessary to understand MDV infection and prevalence in chicken flocks, and to analyze the pathogenic characteristics and virulence variation patterns of wild-type MDV strains in my country. Rapid differential diagnosis of MDV infection and obtaining its genomic information can provide a basis for early detection, control, and source tracing of the virus, minimizing its economic losses to the poultry industry. Nanopore sequencing has the advantages of real-time, portability, long read length, and high throughput, enabling rapid on-site detection. However, nanopore sequencing has certain limitations. For example, compared with first-generation sequencing, second-generation sequencing, and tertiary PACbio sequencing, nanopore sequencing has lower accuracy, especially when the library fragment is too long, the sequencing quality will be severely degraded. Segmented amplification of the genome may solve these problems. However, there is currently no sequencing technology for MDV specifically designed for nanopore sequencing. Therefore, there is an urgent need for a whole-genome sequencing technology for MDV based on third-generation nanopore sequencing to rapidly obtain the pathogen's whole genome sequence, which is of paramount importance for basic research on MDV and its prevention and control. Summary of the Invention
[0004] This invention designs specific primers for long PCR amplification of the entire MDV genome and further provides a real-time MDV detection method using nanopore sequencing, fully leveraging the advantages of nanopore sequencing in pathogen detection, such as real-time processing, portability, long read length, and high throughput. This detection method can rapidly identify and diagnose MDV infection, while simultaneously obtaining its entire genome sequence (including all coding genes and their intergenic regions). The obtained genome sequence can provide information for pathogen variation monitoring and molecular epidemiological studies.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:
[0006] A detection primer set for chicken Marek's virus, comprising 206 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1 to 412.
[0007] The application of the above-mentioned detection primer set in the preparation of products for detecting Marek's virus in chickens.
[0008] Furthermore, the product includes reagents or kits.
[0009] On the other hand, 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; 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.
[0011] On the other hand, 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; 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.
[0013] On the other hand, the present invention provides a method for preparing chicken Marek's virus sequencing fragments, which involves amplifying DNA extracted from the sample to be tested using the above-mentioned detection primer set, and purifying the amplification product to obtain sequencing fragments.
[0014] Furthermore, the DNA obtained from the sample to be tested was amplified by multiplex PCR using the first primer set and the second primer set, respectively, and the amplification products were purified to obtain sequencing fragments.
[0015] 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.
[0016] Furthermore, the sequencing samples are suitable for second- 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] On the other hand, the present invention provides the application of the aforementioned primer set, detection reagent or kit in the non-disease diagnostic detection of Marek's virus in chickens.
[0020] Furthermore, the above-mentioned testing steps include:
[0021] 1) Extract total DNA from the sample.
[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, respectively. The amplification products were purified to obtain sequencing fragments. The first primer set included 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 included 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 amplification products of the two sets of primers were combined and purified to obtain the sequencing fragment.
[0024] 4) Sequencing to obtain genome fragment sequences.
[0025] 5) The genome sequence of chicken Marek's virus was obtained by comparison with and / or assembly of the reference genome.
[0026] Furthermore, the sequencing described 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 from inanimate organisms.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] Based on the publicly available whole genome sequence of MDV in the GenBank gene sequence database of the Center for Biotechnology Information (CBI), 206 pairs of multiplex PCR amplification primers were designed according to genomic locations, as shown in SEQ ID NO. 1-412. The detection method of this invention can obtain the whole genome sequence information of MDV. This invention utilizes the advantages of nanopore sequencing—real-time, portable, long reads, and high throughput—to achieve rapid identification and diagnosis of MDV infection, as fast as 15 hours; simultaneously, it obtains the whole genome sequence, which can provide scientific evidence for virus tracing, pathogen mutation tracking, identification of novel strains, and early warning. Attached Figure Description
[0030] Figure 1 This study shows the sequencing depth coverage of the amplified products from two groups, the 5' end and the 3' end, based on the primer binding position.
[0031] Figure 2 This represents the sequencing depth coverage of two sets of amplified products obtained by randomly combining primer pairs.
[0032] Figure 3 To determine the depth coverage of MinION sequencing for 814 virus strain samples.
[0033] Figure 4 To determine the depth coverage of MinION sequencing for CVI988 strain samples.
[0034] The present invention will be further explained below with reference to specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention. Detailed Implementation
[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art 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 pertains.
[0037] 1. Primer design for MDV third-generation sequencing whole genome amplification
[0038] Based on the publicly available whole genome sequence of *MDV* in the GenBank gene sequence database of the Center for Biotechnology Information (CBI), whole genome multiple sequence alignment analysis was performed using molecular biology tools such as BioEdit to obtain its conserved regions. Primers were designed and validated using Primer3. Multiple primer pairs were designed for whole genome amplification of MDV. Primer information is as follows:
[0039] Table 1. Detection primer sequence involved in this invention.
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046] This detection primer set achieves relatively uniform coverage of MDV, providing a good technical means for obtaining more uniform and effective MDV genome sequencing samples, and has achieved good results in both nanopore sequencing and high-throughput sequencing. This detection primer set is suitable for preparing products for MDV detection and has been widely used. Specifically, it can be a reagent or kit. This invention also protects the reagent or kit, specifically using the above detection primer set as primers to amplify and prepare sequencing fragments of the entire MDV genome.
[0047] 2. Establishing a real-time MDV sequencing method for nanopore sequencing
[0048] This embodiment utilizes the preparation method provided by the present invention to sequence MDV samples. The specific process is as follows:
[0049] 1) Nucleic acid extraction: Nucleic acid was extracted from the sample using a Bori automated nucleic acid extractor. After extraction, the concentration was measured using a Qubit fluorescence quantitative instrument.
[0050] 2) PCR multiplex amplification: The reaction was divided into two parts, tube 1 (first primer set) and tube 2 (second primer set). The same sample was reacted using NEB (NEW ENGLAND BioLabs) High-Fidelity 2X Master Mix under the following conditions: 98℃, 30s; 35 cycles: 98℃, 15s, 65℃, 5min; stored at 4℃.
[0051] 3) PCR product purification: Combine the products from the two amplification pools into a 1.5 ml EP tube. Purify using 0.8× magnetic beads, wash twice with 200 μl of freshly prepared 70% ethanol, and then elute the purified product with nuclease-free water.
[0052] 4) PCR product quality testing: using The HS Assay Kit is a double-stranded DNA fluorescence quantitative reagent kit. Follow the instructions of the quantitative reagent kit to quantify the PCR purified product.
[0053] 5) Third-generation sequencing: The MinION nanopore sequencer was used to construct libraries and sequence mixed samples of amplified products, with an average sequencing data volume of no less than 500×.
[0054] 6) Analysis results: Using bioinformatics methods, the sequencing fragment sequences were aligned to the GenBank reference genome, and the coverage depth of each genomic locus was calculated.
[0055] Experimental Example 1
[0056] The genome was divided into 5' and 3' ends for multiplex PCR amplification. Specifically, primers were divided into 5' end primer set (PCR tube 1) and 3' end primer set (PCR tube 2) according to their binding sites on the PCR sequence (allowing up to 3 mismatches, but not allowed at the 3' end). Taking strain 814 (JF742597) as an example, the 5' end primer set was expected to amplify up to 88043 bp, and the 3' end primer set was expected to amplify from 87933 bp. There was some overlap between the amplification regions of the two primer sets, and other conditions remained unchanged from the above experimental steps.
[0057] The results are as follows Figure 1 As shown, the overall read coverage is extremely uneven, with some sites having coverage of less than 100% or even 0%, making it difficult to meet the requirements for whole-genome coverage.
[0058] Experiment Example 2
[0059] The difference from Experiment 1 is that the primer pairs were randomly combined and divided into two tubes, and only the CVI988 strain (PV035744) sample was sequenced, while other conditions remained the same as the above experimental steps.
[0060] The results are as follows Figure 2 As shown, the overall read coverage is extremely uneven, with some sites having coverage of less than 100% or even 0%, making it difficult to meet the requirements for whole-genome coverage.
[0061] Experimental Example 3
[0062] According to Table 1, the primers are grouped into the first primer group and the second primer group, and the reactions are divided into two groups. Other conditions are carried out according to the above experimental steps.
[0063] The results showed that this amplification system achieved 100% genomic coverage of MDV, with all amplified regions achieving a coverage depth of >100-fold. MinION sequencing results are as follows: Figures 3-4 As shown ( Figure 3 This is a sample of strain 814. Figure 4 (This refers to a CVI988 strain sample). This result demonstrates that the standard evaluation criteria of this invention meet the requirements, providing 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 at appropriate sites, and the primer pair combination needs to be verified to achieve the best results.
[0065] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A primer set for detecting Marek's virus in chickens, characterized in that, The primer set is divided into two groups, each group is used for multiplex PCR. The first primer set consists of SEQ ID NO.1 to SEQ ID NO.208, and the second primer set consists of SEQ ID NO.209 to SEQ ID NO.
412.
2. The application of the detection primer set according to claim 1 in the preparation of products for detecting Marek's virus in chickens.
3. The application according to claim 2, characterized in that, The products include reagents or kits.
4. A detection reagent for Marek's virus in chickens, characterized in that, Includes the detection primer set as described in claim 1.
5. A detection kit for Marek's virus in chickens, characterized in that, Includes the detection primer set as described in claim 1.
6. A method for preparing a sequencing fragment of chicken Marek's virus, characterized in that, The DNA nucleic acid extracted from the sample to be tested is amplified using the detection primer set described in claim 1. The amplification is performed by multiplex PCR using the first primer set and the second primer set respectively. The amplification product is then purified to obtain a sequencing fragment.
7. The primer set of claim 1, the detection reagent of claim 4, or the kit of claim 5 in... Application of non-disease diagnostic testing for Marek's virus in chickens.