Avian leukemia virus whole genome sequencing method based on nanopore sequencing

Abstract

The invention provides an amplification primer and method for an avian leukosis virus whole genome. The detection primer group for the avian leukosis virus comprises 18 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-36. The detection primer group provided by the invention can realize relatively uniform coverage on the avian leukosis virus, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the avian leukosis 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, infection of the avian leukosis virus can be rapidly identified and diagnosed, meanwhile, the whole genome sequence of the avian leukosis 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 invention relates to the technical field of animal virus detection, and in particular to a primer and method for amplifying the whole genome of an avian leukemia virus based on nanopore sequencing. Background Art

[0002] Avian leukosis (AL) is caused by avian leukosis virus (ALV), which is a carcinogenic retrovirus with high infection rate and low morbidity. According to its clinical symptoms and tumor types, it can be divided into lymphocytic leukemia, erythroblastic leukemia, myeloblastic leukemia and myeloid leukemia, among which lymphocytic leukemia is the most common. In the 1950s, avian leukemia was first discovered in Gansu Province, my country, and quickly spread to various provinces. In 1999, ALV-J was first detected in commercial broilers in my country. The etiology showed that it was introduced from white-feathered broiler breeders introduced from abroad. Subsequently, ALV-J infection in white-feathered broilers was reported in Shandong, Jiangsu, Ningxia and other places. With the introduction and hybridization of local chickens in my country, ALV-J has also broken out in many local fine breeds. At present, the main ALV infections in my country are ALV-A, ALV-B and ALV-J, among which ALV-J is the most prevalent in chicken flocks in various places. In recent years, a new subgroup ALV-K has been isolated from local chicken breeds in my country, which has led to increasingly complex ALV infections in my country and brought new challenges to the prevention and control of avian leukosis.

[0003] ALV is a retrovirus, and its replication process is prone to gene mutation and recombination, causing changes in its antigenicity, biological characteristics and pathogenicity. In recent years, ALV has been in rapid mutation and evolution. Rapid differential diagnosis of ALV 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 poultry industry. Nanopore sequencing has the characteristics of real-time, portability, long read length, and high throughput. It can realize real-time and rapid detection on site, timely and accurately identify pathogens, and give accurate detection results. However, there is currently no sequencing technology for ALV designed for the advantages of nanopore sequencing. Therefore, there is still an urgent need for an ALV whole genome sequencing technology based on third-generation nanopore sequencing to quickly obtain the whole genome sequence of the pathogen, 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 ALV, and further provides a real-time detection method of ALV by 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 ALV infection, 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 technical solution adopted by the present invention is:

[0006] In one aspect, the present invention provides a detection primer set for avian leukemia virus, the detection primer set comprising 18 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 36.

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

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

[0009] In another aspect, the present invention provides a detection reagent for avian leukemia virus, comprising the above detection primer set.

[0010] Furthermore, the detection reagent includes a first primer set and a second primer set, wherein the first primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.19 to 36.

[0011] In another aspect, the present invention provides a detection kit for avian leukemia virus, comprising the above detection primer set.

[0012] Furthermore, the detection kit includes a first primer set and a second primer set, wherein the first primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.19 to 36.

[0013] On the other hand, the present invention provides a method for preparing an avian leukemia virus sequencing fragment, wherein the cDNA obtained by reverse transcription of the sample to be detected is amplified using the above detection 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 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 19 to 36.

[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 samples include meconium, egg white, semen or plasma, 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, to perform multiple PCR amplification on the cDNA obtained by reverse transcription of the sample to be tested; wherein the first primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.19 to 36.

[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 avian leukemia virus.

[0024] 5) Obtain the full-length genome sequence of the avian leukemia virus by comparison and / or assembly with the reference genome.

[0025] Furthermore, the non-disease diagnostic test for avian leukemia virus is to detect environmental samples or meconium, egg white and semen or plasma samples of inanimate biological individuals.

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

[0027] According to the full genome sequence of ALV disclosed in the gene sequence database GenBank of the Center for Biotechnology Information of the United States, 18 pairs of amplification primers were designed according to the genome position, and the specific sequences are shown in SEQ ID NO.1 to 36. The detection method of the present invention can obtain the full genome sequence information of ALV. 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 ALV infection, as fast as 15 hours; at the same time, its full 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 ALV-A samples 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 ALV-B samples 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 ALV-J samples 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 ALV-K samples using the comparative primer set in Experimental Example 4 of the present invention.

[0033] Figure 5 This is the depth coverage of MinION sequencing of ALV-A samples using the primer set of the present invention in Experimental Example 5 of the present invention.

[0034] Figure 6 This is the depth coverage of MinION sequencing of ALV-B samples using the primer set of the present invention in Experimental Example 5 of the present invention.

[0035] Figure 7 This is the depth coverage of MinION sequencing of ALV-J samples using the primer set of the present invention in Experimental Example 5 of the present invention.

[0036] Figure 8 This is the depth coverage of MinION sequencing of ALV-K samples using the primer set of the present invention in Experimental Example 5 of the present invention. 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] Table 1 Detection primer set sequences involved in the present invention

[0040]

[0041]

[0042] Example 1 ALV third-generation sequencing whole genome amplification primer design

[0043] According to the whole genome sequence of ALV 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. Multiple pairs of primers were designed for the ALV genome to achieve whole genome amplification. The primer information is shown in Table 1. The detection primer set can achieve relatively uniform coverage of ALV, provide a good technical means for obtaining more uniform and effective ALV 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 ALV 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 ALV.

[0044] Example 2 Establishment of ALV real-time sequencing method using nanopore sequencing

[0045] This example uses the preparation method provided by the present invention to sequence the ALV sample, and the specific process is as follows:

[0046] 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.

[0047] 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℃.

[0048] 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.

[0049] 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.

[0050] 5) PCR product quality inspection: use dsDNA HS Assay Kit: quantify the purified PCR product according to the instructions of the quantification kit.

[0051] 6) Third-generation sequencing: The nanopore sequencer MinION was used to construct a library and sequence the mixed samples of amplified products.

[0052] 7) Analysis results: Bioinformatics methods were used to align the sequencing fragment sequences to the GenBank reference genome and calculate the coverage depth of each genomic site.

[0053] Experimental Example 1

[0054] The genome was divided into 5' and 3' ends for multiplex PCR amplification, specifically

[0055] In step 2), tube 1 of multiplex PCR contains the primer set at the 5' end, SEQ ID NO.1 to SEQ ID NO.4, SEQ ID NO.10 to SEQ ID NO.13, SEQ ID NO.19 to SEQ ID NO.23, and SEQ ID NO.28 to SEQ ID NO.32, and tube 2 contains the remaining primer set, i.e., the primer set at the 3' end. Only ALV-A samples were sequenced, and other conditions remained unchanged according to the above experimental steps.

[0056] 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.

[0057] Experimental Example 2

[0058] The difference from Experimental Example 1 is that the primer pairs are randomly combined and divided equally into 2 tubes, and only the ALV-BV sample is sequenced, and the other conditions remain unchanged according to the above experimental steps.

[0059] 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.

[0060] Experimental Example 3

[0061] The difference from Experimental Example 1 is that the primer pairs are randomly combined and divided equally into 2 tubes, and only the ALV-J sample is sequenced, and the other conditions remain unchanged according to the above experimental steps.

[0062] 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.

[0063] Experimental Example 4

[0064] The difference from Experimental Example 1 is that the primer pairs are randomly combined and divided equally into 2 tubes, and only the ALV-K sample is sequenced, and the other conditions remain unchanged according to the above experimental steps.

[0065] The results are as follows Figure 4 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.

[0066] Experimental Example 5

[0067] 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 5 to 8 As shown, Figure 5 For ALV-A samples, Figure 6 For ALV-B samples, Figure 7 For ALV-J sample, Figure 8 This is the sequencing result of the ALV-K sample.

[0068] The results showed that the amplification system achieved 100% coverage of the ALV 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 ALV genome sequencing samples.

[0069] 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 avian leukosis virus, characterized in that: The detection primer set includes 18 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs. 1 to 36.

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

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

4. A detection reagent for avian leukosis 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 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.19 to 36.

6. A detection kit for avian leukosis 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 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.1 to 18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NOs.19 to 36.

8. A method for preparing a sequencing fragment of avian leukosis 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 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.1~18; the second primer set includes 9 pairs of amplification primers, and the specific sequences are shown in SEQ ID NO.19~36.

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 meconium, egg white, and semen or plasma.

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