Avian leukosis virus whole genome sequencing method based on nanopore sequencing

Abstract

The present invention provides primers and methods for amplifying the entire genome of avian leukosis virus. The detection primer set for avian leukosis virus includes 18 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 36. The detection primer set provided by the present invention can achieve relatively uniform coverage of avian leukosis virus, and in nanopore sequencing, 100% regional coverage of the genome is achieved. The method for preparing sequencing fragments of the entire genome of avian leukosis virus provided by the present invention is simple and easy to operate, with good amplification effect. Nanopore sequencing has the advantage of real-time analysis while sequencing, which can greatly shorten the detection time and can quickly identify and diagnose avian leukosis virus infection. At the same time, its entire genome sequence can be obtained. The obtained genome sequence can provide a scientific basis for virus tracing, pathogen mutation tracking, new strain identification and early warning, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of animal virus detection, and in particular to a primer and method for amplifying the entire genome of an avian leukemia virus based on nanopore sequencing. Background Art

[0002] Avian leukosis (AL) is caused by the avian leukosis virus (ALV), an oncogenic retrovirus characterized by a high infection rate and low morbidity. Based on clinical symptoms and tumor type, AL can be categorized into lymphocytic, erythroblastic, myeloblastic, and myeloid forms, with lymphocytic leukemia being the most common. Avian leukosis was first discovered in Gansu Province, my country, in the 1950s and rapidly spread to various provinces. In 1999, ALV-J was first detected in commercial broiler chickens in my country. Etiological evidence suggests that it was introduced from imported white-feathered broiler breeders. Subsequently, ALV-J infection in white-feathered broiler chickens was reported in Shandong, Jiangsu, Ningxia, and other regions. With the introduction and crossbreeding of local chicken breeds across my country, ALV-J outbreaks have also occurred in many local elite breeds. Currently, ALV-A, ALV-B, and ALV-J are the main strains of ALV infection in my country, with ALV-J being the most prevalent in chicken flocks across the country. In recent years, a new subgroup, ALV-K, has been isolated from local Chinese chicken breeds, complicating the ALV infection landscape and presenting new challenges to avian leukosis prevention and control.

[0003] ALV is a retrovirus, and its replication process is prone to genetic mutation and recombination, causing changes in its antigenicity, biological characteristics and pathogenicity. In recent years, ALV has been in a state of 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 causes 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 accurate identification of pathogens, and provide 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] This study designs specific primers for full-length PCR amplification of the ALV genome and further provides a real-time ALV detection method using nanopore sequencing. This method leverages the advantages of nanopore sequencing for pathogen detection, including real-time, portability, long read length, and high throughput. This method can rapidly identify and diagnose ALV infection and simultaneously obtain the full genome sequence, which 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] In one aspect, the present invention provides a detection primer set for avian leukemia virus, 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-mentioned 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-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 9 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 18; the second primer set includes 9 pairs of amplification primers, the specific sequences of which 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-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 9 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 18; the second primer set includes 9 pairs of amplification primers, the specific sequences of which 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-mentioned detection primer set, and the amplified product is purified to obtain a sequencing fragment.

[0014] Furthermore, 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;

[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.

[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) Multiplex PCR amplification is performed on the cDNA obtained by reverse transcription of the sample to be tested using the first primer set and the second primer set; wherein the first primer set includes 9 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 18; the second primer set includes 9 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 19 to 36.

[0022] 3) The amplified 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 with the reference genome and / or assembly.

[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] Based on the full genome sequence of ALV published in GenBank, the gene sequence database of the Center for Biotechnology Information in the United States, 18 pairs of amplification primers were designed according to genomic location, and the specific sequences are shown in SEQ ID NOs. 1 to 36. The detection method of the present invention can obtain ALV full genome sequence information. The present invention utilizes the advantages of nanopore sequencing in real time, portability, long read length, and high throughput to achieve rapid differential diagnosis of ALV infection in as fast as 15 hours; at the same time, its full genome sequence is obtained. 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 with reference to the accompanying drawings and specific embodiments.

[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 clearly and completely described below in conjunction with the 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 in the technical field 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 examples, unless otherwise specified, are conventional methods. The experimental materials in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores.

[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] Based on the full genome sequence of ALV that has been published 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 ALV genome. The primer information is shown in Table 1. The detection primer set can achieve relatively uniform coverage of ALV, providing a good technical means for obtaining more uniform and effective ALV genome sequencing samples, and has achieved 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 ALV whole genome.

[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 ALV samples. 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, the concentration needs to be measured using Qubit fluorescence quantification instrument.

[0047] 2) First-Strand cDNA Synthesis: Transfer 8 μL of RNA (200 ng / μL) to a new 0.2 mL PCR tube. Add 2 μL of RTMix (TaKaRa) on ice. Gently flick to mix. Centrifuge briefly to remove the liquid from the tube, avoiding any bubbles. Place the PCR tube / plate on ice. Run the PCR cycle in a thermal cycler (25°C for 2 min; 55°C for 10 min; 95°C for 10 min; 4°C hold). Store at 4°C.

[0048] 3) 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.

[0049] 4) PCR product purification: Combine the products from the two amplification pools into a 1.5 mL EP tube. Purify using 0.8× the volume of magnetic beads, rinse twice with 200 μL of freshly prepared 70% ethanol, and elute the purified product with nuclease-free water.

[0050] 5) PCR product quality inspection: use dsDNA HS Assay Kit: Quantify the purified PCR product according to the kit's instructions.

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

[0052] 7) 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.

[0053] Experimental Example 1

[0054] The genome was divided into 5' and 3' ends and multiplex PCR amplification was performed separately.

[0055] In step 2), multiplex PCR tube 1 contains the 5' primer set (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). Tube 2 contains the remaining primer sets (i.e., the 3' primer set). For sequencing only the ALV-A sample, all other conditions remained the same as in the previous 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 were randomly combined and divided equally into two tubes, and only the ALV-BV sample was sequenced. Other conditions remained 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 were randomly combined and divided equally into two tubes, and only the ALV-J sample was sequenced. Other conditions remained 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 were randomly combined and divided equally into two tubes, and only the ALV-K sample was sequenced. Other conditions remained 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, 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 samples, 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 this 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 merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. 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 method for preparing an avian leukosis virus sequencing fragment, characterized in that: The cDNA obtained by reverse transcription of the sample to be tested is amplified using the detection primer set of avian leukosis virus, and the amplified product is purified to obtain a sequencing fragment; The detection primer set 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.

2. The preparation method according to claim 1, characterized in that The sequencing sample is suitable for third-generation sequencing.

3. The preparation method according to claim 1, characterized in that The samples to be tested include clinical samples or environmental samples.

4. The preparation method according to claim 3, characterized in that The clinical samples include meconium, egg white, and semen or plasma.

5. Use of the preparation method according to claim 1 in preparing a product for detecting avian leukosis virus.

6. The use according to claim 5, characterized in that The product includes a reagent or a kit.

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