Primer group, kit and method for amplifying HIV-1 (human immunodeficiency virus-1) genome and application of primer group and kit

Abstract

The invention provides a primer group for targeted amplification of an HIV-1 genome, a kit and a method and application thereof, relates to HIV-1 detection, and belongs to the technical field of genome detection. The universal primer group for amplifying the HIV-1 genome is designed, targeted amplification can be carried out on the HIV-1 genome, sequencing and sequence splicing are carried out by utilizing a three-generation sequencing platform after a library is constructed, and the HIV-1 genome is obtained. The method provided by the invention can amplify all HIV-1 subtypes, and has the advantages of large flux, low cost and simple sequence splicing.

Description

Technical Field

[0001] The present invention belongs to the technical field of genome detection, relates to HIV genome detection, and specifically relates to a primer set, a kit, a method and an application thereof for targeted amplification of the HIV-1 genome. Background Art

[0002] The human immunodeficiency virus (HIV-1) is a retrovirus that is highly susceptible to mutation, with new strains constantly emerging. The lack of proofreading in HIV's reverse transcriptase, the rapid rate of viral replication, and the high frequency of recombination between subtypes contribute to the diversity of the viral genome sequence. This diversity in HIV-1 gene sequences poses significant challenges to global surveillance of HIV-1 molecular prevalence and the diagnosis and treatment of infection. The prevalence of HIV-1 subtypes is regional, with subtypes such as CRF01_AE, B, CRF07_BC, and CRF08_BC predominating in my country.

[0003] The HIV-1 viral genome RNA is approximately 9.7 kb long. The two RNA chains are hydrogen-bonded at the 5' end to form a dimer, with a cap-like structure and a PolyA tail at each of the 5' and 3' ends of the dimer. The structure and assembly of the HIV-1 viral genome are similar to those of other retroviruses, with LTR, gag, pol, env, and LTR arranged in sequence from the 5' end to the 3' end. However, compared to other retroviruses, HIV-1 has more regulatory genes and a more complex genome structure than other retroviruses, hence the name "complex retrovirus." In order to make the most efficient use of the limited genome, the coding regions of HIV-1 genes overlap in many places, especially at the 3' end, where multiple genes appear in the same region.

[0004] Obtaining the complete HIV-1 genome sequence is a crucial foundation for molecular virology research. It plays a crucial role in studying the evolution, transmission, and distribution of the virus within specific populations or regions, identifying new subtypes, prevalent recombinants, or unique recombinants, and developing diagnostic reagents and vaccines. Sequencing remains the most accurate method for identifying viral variants, particularly recombinant viruses. Only full-length sequencing can accurately identify the chimeric pattern of a recombinant virus strain. Currently, commonly used techniques for obtaining viral gene sequences include PCR Sanger sequencing and next-generation sequencing.

[0005] (1) PCR Sanger sequencing: PCR amplification is performed using primers covering the entire genome fragment region, and then Sanger sequencing (first-generation sequencing) is performed on the large number of amplified fragments one by one to obtain the fragment sequences and then assemble them. This technology is cumbersome, labor-intensive, and has a long operation cycle. Taking HIV-1 as an example, the full length of the genome is about 9.7kb. Sanger sequencing can obtain a fragment of about 0.8kb in length in one pass, and obtaining the full length requires more than ten passes of sequencing.

[0006] (2) Second-generation sequencing: It has the characteristics of high sequencing throughput, fast sequencing speed, and high sensitivity. The application of second-generation sequencing technology to deep sequencing of HIV-1 helps to accurately analyze viral quasispecies, detect low-abundance drug-resistant mutations, predict co-receptor tropism, and determine the critical point of drug-resistant mutations that fail ART. In recent years, the second-generation sequencing technology based on metagenomics has emerged. It can detect all viral sequences in the sample without the help of primers or probes, and can detect new viruses. However, it currently faces technical problems such as low data utilization, high detection costs, and the large amount of data required to assemble the genome. If metagenomic sequencing is performed directly from clinical blood samples, there are still a lot of host gene interference problems.

[0007] Third-generation sequencing technology refers to single-molecule sequencing, which is broadly divided into two main categories. The first category is single-molecule fluorescence sequencing, represented by Helicos' SMS technology and Pacific Bioscience's SMRT technology. Deoxynucleotides are fluorescently labeled, and changes in fluorescence intensity can be recorded in real time using a microscope. When the fluorescently labeled deoxynucleotide is incorporated into a DNA strand, its fluorescence is detected simultaneously along the DNA strand. Once the deoxynucleotide forms a chemical bond with the DNA strand, its fluorescent group is cleaved by the DNA polymerase, and the fluorescence disappears. This fluorescently labeled deoxynucleotide does not affect the activity of the DNA polymerase, and after cleavage, the synthesized DNA strand is identical to the natural DNA strand. The second category is nanopore sequencing, represented by Oxford Nanopore in the UK. This novel nanopore sequencing method uses single molecules to pass through a nanopore one by one. Because the nanopore's diameter is extremely small, it only allows a single nucleic acid polymer to pass through. Since individual ATCG bases have different electrical properties, the difference in the electrical signal can be used to identify the type of base that passed through, thus enabling sequencing. As a new generation of sequencing technology, the third-generation sequencing technology has the significant advantage of being able to perform long-fragment sequencing. Therefore, it shows outstanding advantages in whole genome sequencing.

[0008] Prior art CN112080589A discloses a nucleic acid composition and amplification sequencing method for direct amplification of the HIV genome from plasma. This invention employs a three-stage amplification strategy, analyzing and selecting three target fragments for amplification: the first amplicon is the gag-pol amplicon, the second amplicon is the pol-env amplicon, and the third amplicon is the env-nef amplicon. By designing and screening primers targeting these three amplicons, a nearly full-length HIV genome is ultimately obtained. However, this technique has low amplification efficiency, resulting in a low genome coverage of only 75%, and is only applicable to HIV-1 strains B and BC, not all HIV-1 subtypes.

[0009] Prior art CN116656873A discloses a primer set for amplifying the near-full-length genome of HIV 1, and an amplification method using the primer set. The PCR product amplified by this invention can be analyzed by first- and second-generation sequencing technologies to obtain the near-full-length genome sequence of HIV 1, which can be used for subtype identification of HIV 1 strains, drug resistance analysis, and other related research. However, the subsequent sequencing method mentioned in this invention is relatively cumbersome. First-generation sequencing requires segmented sequencing, and more than ten sequencing cycles are required to obtain the full length. Due to the limitation of the length of the sequencing fragment, second-generation sequencing can only break the PCR product into short fragments of about 200bp and then build a library for sequencing. The steps are cumbersome and the latest third-generation sequencing is not applied. Summary of the Invention

[0010] This invention addresses the issues of existing technologies, such as inapplicability to all subtypes, low genome coverage, and cumbersome assembly processes, by providing a primer set, kit, and method and application for targeted amplification of the HIV-1 genome. The primer set provided by this invention can amplify the HIV-1 genome into three large fragments, enabling amplification of all HIV-1 subtypes. Furthermore, sequencing using third-generation sequencing technology provides high genome coverage, high throughput, low cost, and simplified sequence assembly.

[0011] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] In one aspect, the present invention provides a primer set for amplifying the HIV-1 genome, the primer set comprising the nucleic acid sequences shown in SEQ ID NOs. 1-12:

[0013] SEQ ID NO.1: CCTTGAGTGCTHMWAGTRGTTGTGTGCCCRTCTGT;

[0014] SEQ ID NO.2: TTACYTKRATYCCTGSRTAAATYTGACTTGCCCA;

[0015] SEQ ID NO.3: TGAARGAHTGYACTGARAGRCAGCKKAATTTTTTAG;

[0016] SEQ ID NO.4: CAYTTCTCCAATTGTCCYTCA;

[0017] SEQ ID NO.5: WCRGTRCARTGYACACATGG;

[0018] SEQ ID NO.6: CTTKAAGCACTCAARGCAAGCTTTWTTGAGGC;

[0019] SEQ ID NO.7: WKYRGTGTGTGCCCGTCTRTKKTRKGACTC;

[0020] SEQ ID NO.8: YTTYTGTATRTCATTGACAGTCC;

[0021] SEQ ID NO.9: CYDYARYTTCCCTCCARRTCACTCT;

[0022] SEQ ID NO.10: CTCCRATTGTCYYTYATAT;

[0023] SEQ ID NO.11: CTRTTTAAATGGYAGYCTADC;

[0024] SEQ ID NO. 12: TKAAGCACTCAARGCAAGCTTTATKRRGGCTTA.

[0025] To ensure maximum amplification coverage, degenerate bases were introduced into some primers, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents G / C, W represents A / T, H represents A / T / C, and D represents G / A / T.

[0026] In another aspect, the present invention provides a method for amplifying HIV-1 genome, comprising using the above primer set to perform three-segment amplification on the HIV-1 genome.

[0027] Preferably, the method comprises the following steps:

[0028] S1. Obtain HIV-1 RNA from the sample;

[0029] S2, reverse transcription of RNA into cDNA and first amplification;

[0030] S3. Using the amplified product in step S2 as a template, perform a second amplification to obtain a three-fragment product.

[0031] Preferably, the sample in step S1 includes a body fluid sample or an artificial culture sample.

[0032] Preferably, the body fluid sample includes whole blood, serum, plasma, dried blood spots, semen or urine, etc., and the artificial culture sample includes HIV-1 culture cultured in an artificially configured culture medium.

[0033] Specifically, the sample is plasma.

[0034] Preferably, the amplification primers used for the first amplification in step S2 are the primer set shown in SEQ ID NO. 1-6.

[0035] Preferably, the amplification products of the first amplification in step S2 include: amplification product 1, obtained by amplifying SEQ ID NO.1 and SEQ ID NO.2; amplification product 2, obtained by amplifying SEQ ID NO.3 and SEQ ID NO.4; and amplification product 3, obtained by amplifying SEQ ID NO.5 and SEQ ID NO.6.

[0036] Preferably, the amplification primers used for the second amplification in step S3 are the primer set shown in SEQ ID NOs. 7-12.

[0037] Preferably, the amplification products of the second amplification in step S3 include: amplification product a, obtained by amplifying SEQ ID NO.7 and SEQ ID NO.8; amplification product b, obtained by amplifying SEQ ID NO.9 and SEQ ID NO.10; and amplification product c, obtained by amplifying SEQ ID NO.11 and SEQ ID NO.12.

[0038] In another aspect, the present invention provides a method for sequencing the HIV-1 genome, comprising the following steps:

[0039] Amplification products a, b and c were obtained using the above method;

[0040] The amplified products a, b, and c were sequenced to obtain the HIV-1 genome sequence.

[0041] Preferably, the sequencing method is third-generation sequencing. Only full-length sequencing can accurately identify the chimerism pattern of a recombinant virus strain and distinguish the subtype of the virus strain. The advantage of third-generation sequencing is long-fragment sequencing, which can more accurately sequence mutation sites, repetitive structures, and high-GC regions in complex DNA, and sequence splicing is simpler.

[0042] Preferably, the sequencing method is to use a specific third-generation sequencing detector and its supporting reagents and instructions to sequence and assemble the amplification products a, b and c obtained by the above method.

[0043] In another aspect, the present invention provides a kit for amplifying HIV-1 genome, wherein the kit comprises the above primer set.

[0044] Preferably, the kit further comprises a buffer, a reverse transcriptase and a DNA amplification enzyme.

[0045] Preferably, the reverse transcriptase is used for reverse transcription of RNA to obtain cDNA, which is then amplified by PCR.

[0046] Preferably, the reverse transcription reaction system can be integrated with the PCR reaction system into one reaction tube to improve amplification efficiency.

[0047] Preferably, the DNA amplification enzyme is used for PCR amplification.

[0048] Preferably, the buffer is a general term for a variety of buffer solutions, including reverse transcription buffer, first amplification buffer, second amplification buffer and diluents of related reagents, etc.

[0049] In another aspect, the present invention provides use of the above primer set or the above kit in amplifying HIV-1 genome.

[0050] Preferably, the application is a non-disease diagnosis or treatment application, such as obtaining the complete genome sequence of HIV-1 to study the evolution, spread and distribution of the virus in a specific population or region, identify new subtypes or prevalent recombinants, and develop diagnostic reagents and vaccines.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention designs a new universal primer set for targeted amplification of the HIV-1 genome, enabling targeted amplification of the HIV-1 genome. The method provided by this invention can amplify all HIV-1 subtypes, providing high genome coverage, high throughput, low cost, and simplified sequence assembly. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Sequencing read length distribution.

[0054] Figure 2 Phylogenetic tree analysis. DETAILED DESCRIPTION

[0055] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0056] Example 1: Extraction of sample RNA:

[0057] RNA was extracted from clinical plasma samples of HIV-1 patients using the QIAamp Viral RNA Mini kit (Shanghai Qiagen, Cat. No. 52904) according to the kit instructions.

[0058] Example 2: PCR amplification

[0059] To ensure maximum amplification coverage, degenerate bases were introduced into some primers, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents G / C, W represents A / T, H represents A / T / C, and D represents G / A / T.

[0060] 1) One-step RT-PCR amplification

[0061] One-step RT-PCR amplification of the extracted RNA was performed using PrimeScript TM One Step RT-PCR Kit Ver.2 was prepared according to the instructions, see Table 1.

[0062] Table 1 RT-PCR reaction system

[0063] Element Volume (μL) 2x Buffer 10 Enzyme mix 1 Primer mix 1 RNA of Example 1 8

[0064] The reaction procedure is as follows:

[0065] 50°C for 30 min, 94°C for 2 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min / kb;

[0066] The primer sequences are as follows:

[0067] Amplified fragment 1, approximately 2.8 kb in length, forward primer 2.8kF: 5'-CCTTGAGTGCTHMWAGTRGTGTGTGCCCRTCTGT-3' (SEQ ID NO. 1, positions 538-571 in the reference sequence HXB2 (Genbank No. K03455)), reverse primer 2.8kR: 5'-TTACYTKRATYCCTGSRTAAATYTGACTTGCCCA-3' (SEQ ID NO. 2, positions 3378-3345 in the reference sequence HXB2);

[0068] Amplify fragment 2, approximately 5.6 kb in length, using forward primer 5.6kF: 5'-TGAARGAHTGYACTGARAGRCAGGCKAATTTTTTAG-3' (SEQ ID NO. 3, which is at positions 2057-2092 in the reference sequence HXB2), and reverse primer 5.6kR: 5'-CAYTTCTCCAATTGTCCYTCA-3' (SEQ ID NO. 4, which is at positions 7668-7648 in the reference sequence HXB2);

[0069] The amplified fragment three was approximately 2.6 kb in length, using forward primer 2.6kF: 5'-WCRGTRCARTGYACACATGG-3' (SEQ ID NO. 5, at position 6954-6973 in the reference sequence HXB2), and reverse primer 2.6kR: 5'-CTTKAAGCACTCAARGCAAGCTTTWTTGAGGC-3' (SEQ ID NO. 6, at position 9638-9607 in the reference sequence HXB2).

[0070] 2) Slot-type PCR amplification (second round of amplification)

[0071] according to The configuration system of HSDNA Polymerase related reagents is shown in Table 2.

[0072] Table 2 Tank PCR reaction system

[0073] Element Volume (μL) 5x Buffer 4 dNTPs 1 Enzyme 1 Second round primer mix 1 First round PCR products 2 <![CDATA[H2O]]> 11

[0074] The reaction procedure is as follows:

[0075] 35 cycles of 98°C for 10 sec, 60°C for 5 sec, and 72°C for 1 min / kb;

[0076] The primer sequences are as follows:

[0077] Amplified fragment 1, approximately 2.8 kb in length, forward primer 2.8k-2F: 5'-WKYRGTGTGTGCCCGTCTRTKKTRKGACTC-3' (SEQ ID NO. 7, which is at positions 552-581 in the reference sequence HXB2), reverse primer 2.8k-2R: 5'-YTTYTGTATRTCATTGACAGTCC-3' (SEQ ID NO. 8, which is at positions 3326-3304 in the reference sequence HXB2);

[0078] Amplify fragment 2, approximately 5.4 kb in length, using forward primer 5.6k-2F: 5'-CYDYARYTTCCCTCARRTCACTCT-3' (SEQ ID NO. 9, at positions 2243-2266 of the reference sequence HXB2), and reverse primer 5.6k-2R: 5'-CTCCRATTGTCYYTYATAT-3' (SEQ ID NO. 10, at positions 7663-7645 of the reference sequence HXB2);

[0079] The amplified fragment three was approximately 2.6 kb in length, using forward primer 2.6k-2F: 5'-CTRTTAAATGGYAGYCTADC-3' (SEQ ID NO. 11, which is at positions 7002-7021 in the reference sequence HXB2), and reverse primer 2.6k-2R: 5'-TKAAGCACTCAARGCAAGCTTTATKRRGGCTTA-3' (SEQ ID NO. 12, which is at positions 9636-9604 in the reference sequence HXB2).

[0080] Example 3: PCR product purification

[0081] Note: 1. Ampure XP Beads (Becman, Cat. No. A63880) need to be equilibrated at room temperature for more than 30 minutes in advance and thoroughly mixed and resuspended before use.

[0082] 2. 80% ethanol needs to be prepared and used immediately. Do not use reagents that have been stored for too long.

[0083] 1) Combine 20 μL of the second-round three PCR reactions corresponding to each sample into a 1.5 mL Eppendorf EP tube;

[0084] 2) Add 60 μL of resuspended Ampure XP Beads to each EP tube and thoroughly mix the beads and sample by flicking the EP tube;

[0085] 3) Incubate at room temperature for 5 min in a shaking incubator (Eppendorf, model ThermoMixer C);

[0086] 4) Transfer the EP tube to the magnetic stand and let it stand for 2 minutes until the magnetic beads are attracted to one side of the magnetic stand or the mixture becomes clear;

[0087] 5) Carefully discard the supernatant, taking care not to contact the magnetic beads;

[0088] 6) Prepare 80% ethanol using nuclease-free water (Takara Biotech, Cat. No. 9012). Prepare 500 μL of 80% ethanol per sample. Add 200 μL of the prepared 80% ethanol, taking care not to disperse the magnetic beads. After rotating the EP tube or letting it sit for a while, carefully remove and discard the ethanol. Be careful not to touch the magnetic beads during the operation.

[0089] 7) Repeat the washing step once;

[0090] 8) Spin off the sample quickly. Place the sample on a magnetic rack until the beads are completely adsorbed to one side. Carefully discard any remaining liquid with a pipette, avoiding contact with the beads.

[0091] 9) Open the EP tube cap and let it sit for 30s-60s. Do not over-dry.

[0092] 10) Resuspend the precipitated beads in 15 μL of nuclease-free water, flick to mix, incubate at room temperature for 5 minutes, centrifuge briefly, and place on a magnetic rack until the beads are attracted to one side of the rack or the mixture becomes clear.

[0093] 11) Transfer the supernatant to a clean 1.5 mL EP tube, ensuring that no magnetic beads are transferred to the tube;

[0094] 12) Take 1 μL of sample and quantify the DNA concentration using a Qubit fluorometer (Thermo, model Qubit 2.0). If the sample needs to be stored, store it in a -20°C freezer for subsequent experiments.

[0095] Example 4: End Repair

[0096] Prepare the reaction solution according to Table 3 using 200 ng of PCR product from each sample using the NEBNext End repair Module / NEBNext dA-tailing Module kit.

[0097] Table 3 End repair reaction system

[0098] Element Volume (μL) Purified sample of Example 3 12.5 End repair buffer 1.75 End repair enzyme 0.75

[0099] Incubate at 20°C for 20 minutes, 65°C for 10 minutes, and 4°C for at least 1 minute.

[0100] Example 5: Barcode connection

[0101] This example uses the NEB Blunt / TA Ligase Master Mix kit and its instructions.

[0102] 1) Select the corresponding barcode for each sample and add it as the basis for sample identification. A maximum of 24 barcodes can be added simultaneously. The barcode ligation reaction system configuration is shown in Table 4. Mix the reagents by flicking your finger. Use a PCR instrument and incubate at 20°C for 20 minutes, 65°C for 10 minutes, and 4°C for at least 1 minute.

[0103] Table 4 Barcode ligation reaction system

[0104] Element Volume (μL) Example 4 product 7.5 Barcode 2.5 Blunt / TA Ligase Master Mix 10

[0105] 2) Add 2 μL of EDTA to each reaction tube and mix thoroughly to terminate the reaction;

[0106] 3) Pool all incubated samples into a new 1.5 mL Eppendorf DNA LoBind tube and calculate the total volume (Note: For example, for 24 samples, the total volume = 20 * 24 = 480 μL).

[0107] 4) Add 192 μL of AMPure XP beads to 480 μL of barcoded sample, flick gently to mix, and incubate at room temperature for 5 minutes on a shaking incubator (Note: The magnetic bead ratio is 0.4×, and 192 μL is the total amount required for 24 samples. Please calculate the amount for other samples yourself).

[0108] 5) Washing step: Refer to the product purification step in Example 3, and dissolve the sample in 35 μL of nuclease-free water.

[0109] 6) Take 1 μL of sample and quantify the eluted sample using a Qubit fluorometer. The target recovery concentration is approximately 5 ng / μL.

[0110] Example 6: Sequencing Adapter Ligation & Purification

[0111] This example uses the NEBNext Quick Ligation Module kit.

[0112] 1) Reagent Preparation: Thaw SB and SFB at room temperature, vortex to mix, then spin down and place on ice until ready to use. Check each tube for any precipitate. Dissolve AL buffer at room temperature, gently flick to mix AL Ligase and Native Adapter (NA), spin down, and place on ice until ready to use.

[0113] 2) Take the mixed barcoded DNA and ligate the Native Adapter (NA) using the reagents listed in Table 5. Mix the reagents by gently flicking; do not vortex or pipette.

[0114] Table 5 Linker ligation reaction system

[0115] Element Volume (μL) Example 5 Product 30 Native Adapter 5 AL buffer 10 AL Ligase 5

[0116] 3) Mix the sample and centrifuge immediately, then incubate at room temperature for 20 minutes;

[0117] 4) Before use, vortex the AMPure XP beads. Add 20 μL of resuspended AMPure XP beads to the sample, flick gently to mix, and incubate at room temperature on a shaking incubator for 10 minutes.

[0118] 5) Centrifuge the incubated sample tube and place it on a magnetic rack until all the magnetic beads are attracted to one side of the rack and the solution becomes clear and colorless. Aspirate the supernatant, being careful not to touch the magnetic beads.

[0119] 6) Add 125 μL Short Fragment Buffer (SFB), flick the sample to mix, centrifuge briefly, and place on a magnetic rack. Once the beads are completely adsorbed to one side of the magnetic rack and the solution is clear, remove the supernatant.

[0120] 7) Repeat the previous step;

[0121] 8) Place the sample in a centrifuge for a short time, return the EP tube to the magnetic rack, and remove any remaining liquid with a pipette.

[0122] 9) Remove the samples from the magnetic rack and resuspend each sample in 15 μL of Elution Buffer (EB). Incubate at room temperature for 5 minutes.

[0123] 10) Place the EP tube back on the magnetic stand until the eluent becomes clear and colorless;

[0124] 11) Aspirate 15 μL of the clear supernatant and transfer it to a new 1.5 mL EP tube;

[0125] 12) Take 1 μL of sample and quantify the eluted sample using a Qubit fluorometer;

[0126] 13) Place the prepared library on ice for later use.

[0127] Example 7: Library chip loading and sequencing start

[0128] 1) This example uses the UK Oxford Nanopore sequencer M1Kc and its supporting reagents (including SQB, LB and EB, etc.) and instructions. The library is prepared according to Table 6, and about 40ng-50ng of the final prepared library is loaded into the chip.

[0129] Table 6 Composition of the on-machine library

[0130] Element Volume (μl) SQB 37.5 LB 25.5 Library + EB 12

[0131] 2) Add 30 μL Flush Tether (FLT) to a full tube of Flush Buffer (FB) and mix thoroughly to prepare the library loading mixture.

[0132] 3) Open the SpotON sample well on the sequencing chip to remove air bubbles. Pipette 800 μL of library loading solution into the PrimingPort well and let it sit at room temperature for 5 minutes. Rinse and blow-dry the sequencing library suspension to mix thoroughly. Add it dropwise to the SponON sample well. Cover the SpotON sample well and PrimingPort well.

[0133] 4) Install the chip into the sequencer slot, connect the data cable to the computer, start the sequencing control software, select the appropriate sequencer model, chip, reagent kit and other related information parameters from the menu, and start sequencing.

[0134] Example 8: Sequence analysis and assembly

[0135] The fast5 file generated by sequencing in Example 7 was used for base recognition by the Guppy software fast mode that comes with the device, and the current signal was converted into the corresponding base, while the adapter sequence and barcode sequence were removed. After base recognition, it was converted into FASTQ format. Bioinformatics analysis was used to filter sequences with read lengths less than 200bp and longer than the reference genome length (9719bp); sequences with an average sequencing quality value of less than 8 were filtered. The sequencing read length distribution results are as follows: Figure 1 As shown in .

[0136] The filtered sequence was aligned to the reference genome (HXB2, Genbank No.: K03455) and mutation detection was performed, and finally a consensus sequence and VCF format file were generated. The sequencing coverage and sequencing depth of the genome were calculated based on the bam file of the alignment result. The average sequencing depth of the genome obtained by sequencing was 8236.92×, and the coverage greater than 10× was 98.88%. The obtained genome sequence was submitted to NCBI for BLAST analysis. The results showed that the first 100 alignment results displayed were all HIV-1 sequences, among which the highest similarity was with the HIV-1 gene sequence from Uganda with Genbank No. MN650519.1. The results of the phylogenetic tree analysis are as follows Figure 2 shown.

[0137] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A primer set for amplifying HIV-1 genome, characterized in that: The primer set comprises the nucleic acid sequences shown as SEQ ID NO.1-12.

2. A method for amplifying HIV-1 genome, characterized in that: The method comprises using the primer set described in claim 1 to perform three-fragment amplification on the HIV-1 genome.

3. The method according to claim 2, characterized in that The method comprises the following steps: S1. Obtain HIV-1 RNA from the sample; S2, reverse transcription of RNA into cDNA and first amplification; S3. Using the first amplification product in step S2 as a template, perform a second amplification to obtain a three-fragment product.

4. The method according to claim 3, characterized in that: The sample in step S1 includes a body fluid sample or an artificial culture sample.

5. The method according to claim 3, characterized in that: The amplification primers used for the first amplification in step S2 are the primer set shown in SEQ ID NOs. 1-6, and the amplification primers used for the second amplification in step S3 are the primer set shown in SEQ ID NOs. 7-12.

6. A method for HIV-1 genome sequencing, characterized in that: The method comprises the following steps: Using the method according to any one of claims 2 to 5 to obtain a three-fragment product; The obtained fragments were sequenced to obtain the HIV-1 genome sequence.

7. The method according to claim 6, characterized in that The sequencing is third generation sequencing.

8. A kit for amplifying HIV-1 genome, characterized in that: The kit comprises the primer set according to claim 1.

9. The kit according to claim 8, characterized in that The kit further comprises a buffer, a reverse transcriptase or a DNA amplification enzyme.

10. Use of the primer set according to claim 1 or the kit according to any one of claims 8 to 9 in amplifying HIV-1 genome, characterized in that: The application is non-disease diagnosis or treatment application.

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