Capture primer group, kit and capture method for HIV-1 whole genome sequencing and application of capture primer group, kit and capture method

Abstract

The invention provides a capture primer group for HIV-1 whole genome sequencing, a kit, a capture method and application thereof, 31 pairs of imbricate amplification primers are designed according to the imbricate principle for the whole genome of HIV-1, and the 31 pairs of primers are divided into two primer pools, so that HIV-1 whole genome reverse transcription and capture amplification can be performed on RNA extracted from plasma, and the detection sensitivity is high. The obtained DNA product is subjected to a second-generation or third-generation whole genome sequencing experiment to obtain the whole gene of the HIV-1, and the method has the advantages of convenience and rapidness, and is convenient for meeting current molecular network propagation mode analysis, virus recombination variation and traceability research.

Description

Technical Field

[0001] The present invention belongs to the technical field of genome sequencing, and in particular relates to a capture primer set, a kit, a capture method and applications thereof for HIV-1 whole genome sequencing. Background Art

[0002] AIDS, or acquired immune deficiency syndrome (AIDS), is a chronic and fatal infectious disease caused by the human immunodeficiency virus (HIV). It is contagious and highly lethal, and has become one of the major diseases that need to be prevented and controlled worldwide.

[0003] HIV, which is prevalent worldwide, can be divided into HIV-1 and HIV-2 based on differences in viral gene sequences. Currently, HIV-1 is the main prevalent virus in the world. The HIV-1 genome consists of approximately 9.7kb of RNA, including nine different genes that can encode 15 proteins. There are three main genes in its genome: gag, pol, and env. Among them, the gag gene encodes the structural protein of the viral nucleocapsid, the pol gene encodes the enzyme involved in viral replication, and the env gene encodes the viral envelope glycoprotein. After HIV-1 enters the human body, it selectively invades the CD4+T lymphocytes and macrophages of the human immune system, synthesizes cDNA through the reverse transcription mechanism in the cell, forms a pre-integration complex, and integrates into the genomic DNA of the host cell. The main transmission methods of HIV-1 include mother-to-child transmission, sexual contact transmission, blood transmission, etc., which can cause damage to the human immune function. There is currently no drug that can cure AIDS or a preventable vaccine.

[0004] HIV whole genome sequencing technology can detect drug-resistant mutations of the virus, predict the sensitivity of the virus to different drugs, analyze the characteristics of the molecular transmission network, and track the transmission route and source of the virus. At present, conventional HIV-1 detection methods include antibodies, western blot detection, first-generation sequencing of gene fragments and high-throughput sequencing technology, and NGS detection methods for major drug resistance sites related to commonly used antiviral drugs in my country and inferior drug-resistant strains in sensitive HIV-1 primary infections. However, these methods cannot quickly and efficiently obtain the whole genome information of the virus, and thus cannot meet the current molecular network transmission pattern analysis, virus recombination mutation and traceability research. Summary of the invention

[0005] In view of this, the present invention provides a capture primer set, a kit, a capture method and applications thereof for HIV-1 whole genome sequencing, aiming to quickly and efficiently achieve HIV-1 whole genome sequencing, thereby meeting the current molecular network transmission pattern analysis, virus recombination mutation and traceability research.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] A capture primer set for HIV-1 whole genome sequencing, comprising 31 pairs of shingled amplification primers designed for the HIV-1 whole gene, wherein the sequences of the 31 pairs of shingled amplification primers are shown in SEQ ID No. 1-62 in sequence; wherein 15 pairs of even-numbered primers constitute a first primer pool, and 16 pairs of odd-numbered primers constitute a second primer pool, wherein the first primer pool is used to amplify HIV-1 to obtain a first product, and the second primer pool is used to amplify HIV-1 to obtain a second product, and the HIV-1 whole gene fragment is obtained by combining the first product and the second product.

[0008] Preferably, the amplification length of each pair of shingled amplification primers is distributed in the range of 367-453 bp, and the overlapping region of the amplification products of each pair of adjacent primers is in the range of 57-118 bp.

[0009] A kit for HIV-1 whole genome sequencing comprises the capture primer set for HIV-1 whole genome sequencing.

[0010] A method for capturing the whole genome sequence of HIV-1, comprising the following specific steps:

[0011] (1) RNA extraction: extracting RNA from HIV-1 virus in the sample to be tested;

[0012] (2) RNA reverse transcription: The extracted viral RNA is reverse transcribed to generate cDNA chains;

[0013] (3) Primer design and capture amplification: 31 pairs of shingled amplification primers are designed for the whole HIV-1 gene. The sequences of the 31 pairs of shingled amplification primers are shown in SEQ ID No. 1-62, wherein 15 pairs of even-numbered primers constitute a first primer pool, and 16 pairs of odd-numbered primers constitute a second primer pool; using a PCR amplification system, taking the cDNA chain synthesized in step 2, and using primers in the first primer pool and primers in the second primer pool to perform multiple PCR amplification, respectively, to obtain a first product and a second product;

[0014] (4) Sequencing: The first product and the second product are combined and purified to construct a library, which is then sequenced to obtain the HIV-1 full genome sequence; the method for capturing the HIV-1 full genome sequence is a non-disease diagnostic method.

[0015] Preferably, in step (1), the sample to be tested is plasma.

[0016] Preferably, in step (4), sequencing adopts a second-generation whole-genome sequencing method or a third-generation whole-genome sequencing method.

[0017] The capture primer set is used in preparing products for HIV-1 viral genome sequencing.

[0018] It can be seen from the above technical scheme that the present invention provides a capture primer set, a kit, a capture method and its application for whole genome sequencing of HIV-1. Compared with the prior art, the beneficial effects are as follows: this scheme is aimed at the whole genome of HIV-1, and 31 pairs of shingled amplification primers are designed according to the shingled principle to ensure the specificity and sensitivity of the primers, and the 31 pairs of primers are divided into a first primer pool and a second primer pool, respectively, for reverse transcription and capture amplification of the whole genome of HIV-1, so that the whole genome sequence of HIV-1 can be obtained after the two amplified products are mixed and sequenced, simplifying the sample processing process. This scheme can be used for non-diagnostic purposes to reverse transcribe and enrich and amplify the whole genome of HIV-1 from various nucleic acid samples. Its advantages are convenience and rapidity, which is convenient to meet the current molecular network transmission mode analysis, virus recombination mutation and traceability research. In addition, the capture primer set of this scheme can realize the whole genome amplification of all types of HIV-1, and can be applied to all mainstream sequencing platforms of the second or third generation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the design diagram of whole genome capture primers.

[0020] Figure 2 This is the sample S1 data analysis.

[0021] Figure 3 This is the sample S2 data analysis.

[0022] Figure 4 It is a sample S3 data analysis. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1 HIV-1 shingled PCR primer design

[0025] The present invention specifically uses multiplex PCR technology, and a key point of multiplex PCR technology is to design a reasonable multiplex PCR primer pair combination, ensure that each pair of primers will not interfere with each other during amplification, and need to cover the whole genome of HIV-1, and ensure that all typing can be successfully amplified. The present invention uses the NCBI database to obtain the whole genome sequence of various genotypes of HIV-1, and designs 31 pairs of shingled amplification primers according to the shingled principle through the conservative segment, so that the PCR product has an overlapping sequence part, and then the amplification position of the primer is more accurate, thereby improving the specificity and sensitivity of PCR, and because the HIV-1 genome is long, the genome can be divided into multiple overlapping fragments for amplification using shingled PCR, avoiding non-specific products and false positive results generated during PCR amplification, and improving the accuracy of sequencing; and the 31 pairs of primers are divided into a first primer pool and a second primer pool, respectively, and the whole genome of HIV-1 is reverse transcribed and captured and amplified, so that the two amplified products are mixed and the whole genome sequence of HIV-1 is obtained through sequencing experiments, simplifying the sample processing process. The sequences of the 31 pairs of shingled amplification primers are shown in SEQ ID No.1-62 respectively; among them, 15 pairs of even-numbered primers constitute a first primer pool, and 16 pairs of odd-numbered primers constitute a second primer pool. The first primer pool is used to amplify HIV-1 to obtain a first product, and the second primer pool is used to amplify HIV-1 to obtain a second product. The HIV-1 full gene fragment is obtained by combining the first product and the second product.

[0026] Table 1 below shows the primer sequences in the first primer pool and the second primer pool:

[0027] Table 1 Capture primer sequence list

[0028]

[0029]

[0030]

[0031] Example 2: Library construction and sequencing based on HIV-1 whole genome capture method

[0032] The specific operations for library construction and sequencing based on the HIV-1 whole genome capture method are as follows:

[0033] 1. RNA extraction: Take three plasma samples of HIV-1 infection confirmed by Ningxia CDC, and extract viral RNA from plasma according to the instructions of the commercial nucleic acid extraction and purification kit of Tianlong Technology Co., Ltd.;

[0034] 2. RNA reverse transcription: The extracted viral RNA is reverse transcribed to generate a cDNA chain; reverse transcription is performed according to the reaction procedure shown in Table 2 below to generate a cDNA chain:

[0035] Table 2 Reverse transcription system

[0036] Reagents volume Random hexamers 1μL dNTP Mix 1μL RNA template 10μL Total volume 12μL

[0037] Place the PCR tube in a PCR instrument and perform reverse transcription. The PCR execution program is as shown in Table 3:

[0038] Table 3 Reverse transcription procedure

[0039]

[0040]

[0041] 3. Primer design and capture amplification: A whole genome capture system was used to perform PCR amplification on the sample using primers in the first primer pool and the second primer pool, respectively, to obtain the first product and the second product; the whole genome capture system is shown in Table 4, and the PCR capture amplification procedure is shown in Table 5:

[0042] Table 4 Whole genome capture system

[0043] Reagents volume Reaction solution 25μL First primer pool / Second primer pool 3-5μL Enzymes 2μL Reverse transcription product 5μL ddH2O 15μL Total volume 50μL

[0044] Table 5 PCR capture amplification program

[0045] step temperature time Cycle times 1 98℃ 30s 2 98℃ 15s 3 65℃ 4min 25 times 4 72 5 1 time

[0046] 4. Sequencing: The two tubes of amplified product mixture were purified with 1 volume of magnetic beads. After purification, samples with Qubit quantification above 10 ng / ul could be directly used for subsequent library construction experiments. The library construction was carried out according to the manufacturer's instructions with a starting amount of 0.5 ng DNA for each sample. The purified PCR product was quantified using the Qubit dsDNA BR Assay kit (Thermo Fisher Scientific), and the purified library was sequenced using the Miseq sequencer. The fastq files after sequencing were uploaded to the CLC Genomics Workbench for analysis.

[0047] The data of library construction experiment and machine conditions are shown in Table 6. The results of data analysis after sequencing are shown in Table 7. It can be seen that the sequencing quality Q30 of the three samples is about 90%, among which the sequencing of samples 2 and 3 requires less data, with a single sample of <1M reads, which is suitable for all mainstream second-generation or third-generation sequencing platforms.

[0048] Table 6 Library construction experiment and machine condition data

[0049]

[0050] Table 7 Sequencing data analysis

[0051] Sample No. Sequencing quality Q30 (%) Sequencing reads Number of sequencing bases S1 91.3 1.97M 355.90Mb S2 90.6 0.22M 310.60Mb S3 89.7 0.14M 210.09Mb

[0052] Figure 2 , Figure 3 , Figure 4 Table 8 shows the whole genome sequence coverage distribution of the three samples in this embodiment. Table 8 shows the sample sequence length, coverage and reference sequence detailed data. It can be seen that the whole genome sequencing results of the three samples cover more than 95% of the genome.

[0053] Table 8 Sample sequencing analysis results

[0054] Sample No. Covering length Coverage Reference sequence S1 9306 95.8％ KC503855.1 S2 9578 98.6％ KC492738.1 S3 9442 97.2％ OQ513527.1

[0055] What is disclosed above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A capture primer set for whole genome sequencing of HIV-1, characterized in that: It includes 31 pairs of shingled amplification primers designed for the whole HIV-1 gene, and the sequences of the 31 pairs of shingled amplification primers are shown in SEQ ID No.1-62 in sequence; wherein, 15 pairs of even-numbered primers constitute a first primer pool, and 16 pairs of odd-numbered primers constitute a second primer pool, the first primer pool is used to amplify HIV-1 to obtain a first product, and the second primer pool is used to amplify HIV-1 to obtain a second product, and the whole HIV-1 gene fragment is obtained by combining the first product and the second product.

2. The capture primer set for HIV-1 whole genome sequencing according to claim 1, characterized in that: The amplification length of each pair of shingled amplification primers is distributed in the range of 367-453 bp, and the overlapping region of the amplification products of each pair of adjacent primers is in the range of 57-118 bp.

3. A kit for HIV-1 whole genome sequencing, comprising the capture primer set for HIV-1 whole genome sequencing as claimed in claim 1.

4. A method for capturing the complete genome sequence of HIV-1, characterized in that: The specific steps include: (1) RNA extraction: Extract RNA from HIV-1 virus in the sample to be tested; (2) RNA reverse transcription: reverse transcription of the extracted viral RNA to generate cDNA chain; (3) Primer design and capture amplification: 31 pairs of shingled amplification primers are designed for the whole HIV-1 gene. The sequences of the 31 pairs of shingled amplification primers are shown in SEQ ID No. 1-62, wherein 15 pairs of even-numbered primers constitute a first primer pool and 16 pairs of odd-numbered primers constitute a second primer pool. Using a PCR amplification system, the cDNA chain synthesized in step 2 is taken and multiplex PCR amplification is performed using primers in the first primer pool and primers in the second primer pool, respectively, to obtain a first product and a second product. (4) Sequencing: The first product and the second product are combined and purified to construct a library, which is then sequenced to obtain the HIV-1 full genome sequence; the method for capturing the HIV-1 full genome sequence is a non-disease diagnostic method.

5. The method for capturing the complete genome sequence of HIV-1 according to claim 4, characterized in that: In the step (1), the sample to be tested is plasma.

6. The method for capturing the complete genome sequence of HIV-1 according to claim 4, characterized in that: In the step (4), sequencing is performed using a second-generation whole-genome sequencing method or a third-generation whole-genome sequencing method.

7. Use of the capture primer set for HIV-1 whole genome sequencing as claimed in claim 1 in preparing a product for HIV-1 viral genome sequencing.

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