HIV-1 drug resistance gene detection method based on NGS, kit and application

Abstract

The invention belongs to the technical field of detection, and particularly discloses an HIV-1 drug resistance gene detection method based on NGS, a kit and application. The detection method comprises the following steps: S1, extracting RNA of an HIV-1 serum or plasma sample; s2, amplification and enrichment of a drug resistance detection region: amplifying a protease region, a reverse transcriptase region and an integrase region of the HIV-1 POL gene through one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and detecting amplification products; s3, library construction: performing fragmentation, linker addition and tagging synchronous enrichment operation on an amplified product in S2 to construct a sequencing library; s4, sequencing on a machine; s5, data analysis: according to the data sequenced in the step S4, performing analysis to obtain genotypes and drug resistance evaluation of HIV-1 POL gene protease, a reverse transcriptase region and an integrase region. According to the invention, only one-step RT-PCR is needed, and the HIV-1 POL gene drug resistance detection area can be specifically enriched without two rounds of nested PCR.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a method, a kit and an application of detecting HIV-1 drug resistance genes based on NGS. Background Art

[0002] Acquired immune deficiency syndrome (AIDS) is caused by infection with the retrovirus HIV. The source of infection is HIV-infected people and AIDS patients. HIV mainly exists in the body fluids of the source of infection, such as blood, semen, vaginal secretions, pleural effusion, cerebrospinal fluid, amniotic fluid and breast milk. It is transmitted through sexual contact, blood and blood products, and mother-to-child transmission.

[0003] HIV is divided into HIV-1 and HIV-2. HIV-1 is prevalent worldwide and can be divided into four groups: M, N, O, and P. The most important M group can be divided into AD, FH, J, and K subtypes. N, O, and P group strains are mainly found in Central Africa and have a low prevalence rate. HIV-2 is mainly found in Africa and has more than 7 subtypes, including AG. The HIV genome is a single-stranded RNA with about 9,000 bases, containing 9 genes and encoding a total of 15 proteins. Among them, the polymerase (pol) gene encodes a variety of viral enzymes, including proteases, reverse transcriptases, and integrases.

[0004] Antiretroviral therapy (ART) is an effective means to reduce the mortality of HIV-infected people and reduce the spread of HIV. However, due to the high-speed replication of HIV itself and the lack of self-proofreading function during replication, HIV is prone to gene mutation and drug resistance. At the same time, under the selective pressure of long-term antiviral drugs, the probability of HIV drug resistance mutation is greatly increased. The generation and development of drug resistance mutations will affect the efficacy of antiviral treatment. Therefore, timely drug resistance detection plays an important role in the prevention and treatment of AIDS.

[0005] The Sanger sequencing detection method is still the gold standard for clinical detection of HIV drug resistance. First, the protease, reverse transcriptase and / or integrase regions of the POL gene are obtained through RT-PCR, and then Sanger sequencing is performed to obtain sequence information, which is compared with the reference nucleic acid sequence to evaluate whether the drug resistance site has mutated. The Sanger sequencing method has some technical disadvantages, mainly low sensitivity, poor detection effect for mutations below 20% or even higher abundance, and subjective interpretation of results.

[0006] In view of the shortcomings of Sanger sequencing, the method of detecting drug-resistant mutations based on deep sequencing (Next-generation sequencing, NGS) has solved this problem and can even detect mutations below 1%. However, the existing methods for detecting HIV-1 RNA POL gene resistance have the following shortcomings: 1. The detection area is not fully covered. It does not cover the HIV-1 RNA POL gene protease, reverse transcriptase and integrase regions at the same time, and cannot meet the detection area requirements recommended by the latest guidelines; 2. Capturing and enriching the drug-resistant region of HIV-1 RNA POL gene involves two rounds of PCR, one of which is to first perform RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and then perform two rounds of nested PCR amplification and enrichment. The other is to first perform reverse transcription (Reverse Transcription) to synthesize cDNA, and then perform one round of PCR and two rounds of nested PCR amplification and enrichment. The enriched product is subjected to first-generation sequencing or second-generation sequencing detection and analysis. It is impossible to achieve high sensitivity and high specificity in capturing and enriching the protease, reverse transcriptase and integrase resistance detection areas in one round of RT-PCR. The operation is cumbersome and time-consuming. At the same time, the operation of opening the lid and transferring the tube has the risk of aerosol cross-contamination. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a detection method, kit and application of one-step RT-PCR based on NGS for detecting HIV-1 drug resistance genes. Specific primers are used to perform RT-PCR in a one-step method to amplify and enrich the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene, and the library is built by combining the transposase method with the second-generation sequencing, on-machine sequencing and bioinformatics analysis or online database analysis to quickly obtain the genotypes of the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene, and evaluate HIV-1 drug resistance.

[0008] The technical solution adopted by the present invention is as follows: <First aspect> A method for detecting HIV-1 drug resistance genes based on NGS, comprising the following steps: S1. Extract RNA from HIV-1 serum or plasma samples; S2, amplification and enrichment of drug resistance detection regions: one-step RT-PCR amplification of the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene, and detection of the amplified products; S3, library construction: fragmentation, linker addition, and tagging of the amplified products of S2 are performed to construct a sequencing library; S4, sequencing on the machine; S5. Data analysis: Based on the sequencing data in step S4, the genotypes of the protease, reverse transcriptase, and integrase regions of the HIV-1 POL gene and the drug resistance assessment are obtained.

[0009] In step S2, PooL1 is amplified by primer pair PooL1_F_1 and PooL1_R_1; PooL2 is amplified by primer pair PooL2_F_1 and PooL2_R_1; PooL1 (protease and part of the reverse transcriptase region (reference sequence OQ747186.1: 2074-3632) and PooL2 (part of the reverse transcriptase and integrase region (reference sequence OQ747186.1: 3324-4942)) constitute the HIV-1 drug resistance gene region including the protease region, reverse transcriptase region and integrase region of the POL gene; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4.

[0010] Usually, serum samples are extracted immediately or stored at -80°C after separation; plasma sample sampling requires fresh EDTA-anticoagulated whole blood, and it is recommended to separate plasma within 2 hours and no more than 6 hours; the HIV-1 RNA virus sample concentration is not less than 500 copies / mL, the extracted plasma sample is >= 200μL, and the total RNA volume is about 50 µL. High-concentration samples can be diluted before use to avoid repeated freezing and thawing.

[0011] In step S2, the specific primer design process involves reference sequence selection, sequence analysis and primer design process: download all HIV-1 complete genome sequences from the NCBI database, retain genome sequences with a length of not less than 98% of the reference genome NC_001802.1 sequence, filter low-quality sequences (including uncertain bases N greater than 10), remove duplicate sequences with a sequence similarity of more than 99%, and the remaining are representative reference sequences. Use bioinformatics software such as mafft or Clustal Omega or offline bioinformatics analysis processes to perform multiple sequence alignment (Multiple Sequence Alignment) on the remaining representative reference sequences, identify conservative sequences from multiple related sequences, select the most representative reference sequence of the target region, and then use primer3 or other primer design software to design amplification primers for the most representative reference sequence target region sequence. After the amplification primers are designed, check the coverage of the primer sequence and the representative reference sequence, appropriately increase some merged bases, and improve the inclusiveness of the primer sequence to different reference sequences.

[0012] In step S2, the RT-PCR reaction system includes: reverse transcriptase, DNA polymerase, dNTPs, buffer and amplification primers.

[0013] After mixing high-concentration reverse transcriptase, DNA polymerase, dNTPs, and 5X buffer, add appropriate amount of nuclease-free water to make 2 X One Step RT-PCR master mix; the concentrations of each component of 2 X One Step RT-PCR master mix are dNTPs 5mM, reverse transcriptase 5U / μL, DNA polymerase 0.08U / μL, and 2X buffer.

[0014] The reverse transcriptase includes: common commercially available MMLV reverse transcriptase; preferably RNase H-MMLV reverse transcriptase. The 5X buffer contains: 125 mM Tris-HCl (pH 8.3), 250 mM KCl, 15 mM MgCl2, 50 mM (NH4)2SO4, 50 mM DTT, and RNase Inhibitor 5U / μL.

[0015] The DNA polymerase is: a common commercially available DNA polymerase, including but not limited to Taq DNA polymerase, PfuDNA polymerase, Q5® High-Fidelity DNA Polymerase, etc. Ordinary Taq DNA polymerase can achieve the desired effect.

[0016] The amplification primers include: a primer pair PooL1_F_1 and PooL1_R_1 for amplifying PooL1; and a primer pair PooL2_F_1 and PooL2_R_1 for amplifying PooL2; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4; The final reaction concentration of PooL1_F_1 and PooL1_R_1 is 0.1~1μM, preferably 0.4μM.

[0017] The final reaction concentration of PooL2_F_1 and PooL2_R_1 is 0.1~1μM, preferably 0.4μM.

[0018] The RT-PCR reaction procedure includes the following: Reverse transcription: temperature 42℃~55℃, time 5~40 minutes, 1 cycle; cDNA pre-denaturation: temperature 94℃~98℃, time 10~300 seconds, 1 cycle; Denaturation, temperature 94℃~98℃, time 10~60 seconds, annealing, temperature 56~66℃, time 20~60 seconds, extension, temperature 72℃, time 30~300 seconds, 35~50 cycles; Final extension, 68℃~72℃, time is 2~20 minutes, 1 cycle.

[0019] Further, the RT-PCR reaction procedure includes the following: Reverse transcription: temperature 50°C, time 30 minutes, 1 cycle; cDNA pre-denaturation: temperature 95°C, time 180 seconds, 1 cycle; Denaturation, temperature is 95℃, time is 20 seconds, annealing, temperature is 60℃, time is 45 seconds, extension, temperature is 72℃, time is 120 seconds, 42~48 cycles.

[0020] Final extension, 72°C, 5 minutes, 1 cycle.

[0021] Step S2 also includes a step of purifying the amplified product.

[0022] The purification is performed by magnetic bead purification, and the ratio of magnetic beads is 0.5X~2.0X, preferably 0.8X.

[0023] Step S3, constructing a sequencing library includes the construction method of Illumina, MGI library, or Ion Torrent library.

[0024] In step S3, the amount of DNA sample input is 0.1ng~100ng, and Tagment-Beads (transposase coupled to magnetic beads) is preferably used for fragmentation and linker addition. Stop buffer is added to the fragmented product to terminate the reaction, and then the amplification enzyme system and N5XX and N7XX tag primers are added for amplification. The amplification is performed for 9~14 cycles, and the product is purified by magnetic beads. The purified product is subjected to quality inspection and pooling to become the library to be sequenced.

[0025] In S3, the steps of fragmentation, linker addition and tagging simultaneous enrichment operations include: 1) DNA fragmentation and adapter addition: Prepare a reaction system containing 5× reaction buffer, tagment beads, DNA and ddH2O, mix well and place in a PCR instrument, react at a specific temperature and time to fragment the DNA and add adapters; 2) Stop the fragmentation reaction: After the reaction is finished, take out the sample, add Stop buffer, mix well, put it back into the PCR instrument for incubation, and then take it out; 3) Index PCR amplification: Using the fragmented product as a template, prepare a reaction system containing the fragmented product, Ampli Mix, and N5XX + N7XX index primers. Mix and centrifuge, then place in PCR for amplification. Finally, react at a specific temperature and time, purify the reaction product, and obtain the corresponding library.

[0026] The target detection area is fragmented, adapters are added, tags are added for simultaneous enrichment, and sequencing libraries are constructed. Any library construction method can be selected. After the quality inspection is completed, different libraries are mixed by pooling, diluted, denatured to the concentration for use on the machine.

[0027] The primers for PooL1 are selected from the primer set PooL1_1, and the primers for PooL2 are selected from the primer set PooL2_1, see the table below:

[0028] Among them, R and Y are substituted bases.

[0029] <Second Aspect> The present invention also provides a specific primer combination for one-step RT-PCR amplification of the protease region, reverse transcriptase region and integrase region of HIV-1 POL gene, comprising the following: Primer pair PooL1_F_1 and PooL1_R_1 for amplification of PooL1, and primer pair PooL2_F_1 and PooL2_R_1 for amplification of PooL2; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4; The PooL1 and PooL2 constitute the HIV-1 drug resistance gene region including the POL gene protease region, reverse transcriptase region and integrase region.

[0030] <Third Aspect> The present invention also provides a kit comprising the primer combination as described above.

[0031] <Fourth Aspect> The present invention also provides a detection method, primer combination, or kit as described above for use in HIV-1 drug resistance monitoring, prognosis evaluation, epidemiological research, or drug development.

[0032] <Fifth Aspect> The present invention also provides a detection system for HIV-1 drug resistance genes based on NGS, characterized in that it comprises: A sample processing module for extracting RNA from HIV-1 serum or plasma samples; an amplification module for one-step RT-PCR amplification of the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene; The library construction module is used to perform fragmentation, adapter addition, and tagging of the amplified products for simultaneous enrichment operations; A sequencing module is used to perform sequencing on the constructed sequencing library; The data analysis module is used to analyze sequencing data to obtain the genotype and drug resistance evaluation of the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene.

[0033] The amplification module uses a specific primer combination to amplify the protease region, reverse transcriptase region and integrase region of the HIV-1 POL gene; the specific primer combination includes: Primer pair PooL1_F_1 and PooL1_R_1 for amplification of PooL1, and primer pair PooL2_F_1 and PooL2_R_1 for amplification of PooL2; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4; The PooL1 and PooL2 constitute the HIV-1 drug resistance gene region including the POL gene protease region, reverse transcriptase region and integrase region.

[0034] The library construction module uses the transposase method to perform fragmentation, linker addition and tagging operations.

[0035] The data analysis module obtains the genotype and drug resistance evaluation of the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene through bioinformatics analysis or online database analysis.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. Only one-step RT-PCR is needed, without two rounds of nested PCR, to enrich the HIV-1 POL gene drug resistance detection region with high sensitivity and specificity; 2. The magnetic bead-coupled transposase method is used for library construction. No homogenization is required. The DNA input amount ranges from 0.1 to 100 ng and can be directly used for downstream library construction, with a wide compatibility range.

[0037] 3. It has the advantages of simple operation, short time consumption, high sensitivity, strong specificity, full detection area, low risk of cross contamination, low cost and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 The present invention is a flow chart of the method for detecting HIV-1 drug resistance genes based on NGS; Figure 2 The PCR amplification product fragment analysis results of sample 1 in Example 3; Figure 3 The PCR amplification product fragment analysis results of sample 2 in Example 3; Figure 4 The PCR amplification product fragment analysis results of sample 3 in Example 3; Figure 5 The PCR amplification product fragment analysis results of sample 4 in Example 3; Figure 6This is the fragment analysis diagram of the sample 1 library in Example 3; Figure 7 This is a fragment analysis diagram of the sample 2 library in Example 3; Figure 8 This is a fragment analysis diagram of the sample 3 library in Example 3; Fig. 9 This is the fragment analysis diagram of the sample 4 library in Example 3. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0040] Example 1 Figure 1 This is a flow chart of the detection method of HIV-1 drug resistance gene based on NGS of the present invention; This example is used to illustrate the acquisition of target fragments of HIV-1 POL gene protease, reverse transcriptase region and integrase region 1. Sample extraction: HIV-1 RNA samples were extracted from serum or plasma samples of AIDS patients (serum samples were used in this embodiment). First, 1600~2000 xg (2000 xg was used in this embodiment), 4°C, centrifuged for 10~20min (centrifuged for 15min in this embodiment) to separate plasma or serum, and then extracted immediately or stored at -80°C. Then, QIAGEN Viral RNAMini Kit (Cat. No.: 52904 / 52906) or SuoShi Bio Virus Extraction Kit (Magnetic Bead Method) (Cat. No.: SDK60104) was used, and the specific instructions were followed.

[0041] 2. Specific RT-PCR amplification: HIV-1 specific RT-PCR primers were used to amplify the RNA extracted in step (1). The primer set was as shown in Table 1: Table 1

[0042] Among them, R and Y are fused bases.

[0043] The one-step RT-PCR amplification reaction system is shown in Table 2: Table 2

[0044] Among them, the amplification primers in PooL1 are PooL1_F_1 and PooL1_R_1, the primer working solution concentration is 10μM, and the final concentration is 0.4μM; the amplification primers in PooL2 are PooL2_F_1 and PooL2_R_1, the primer working solution concentration is 10μM, and the final concentration is 0.4μM.

[0045] The concentrations of the components of 2X One Step RT-PCR master mix are as follows: 5 mM dNTPs, 5 U / μL RNase H-MMLV reverse transcriptase, 0.08 U / μL TaqDNA polymerase, and 2X buffer.

[0046] Among them, 2X One Step RT-PCR master mix is ​​prepared by mixing 100mM dNTPs: 50U / μL RNase H-MMLV reverse transcriptase: 4 U / μL Taq DNA polymerase: 5X buffer (containing 125mM Tris-HCl (pH 8.3), 250mM KCl, 15mM MgCl2, 50mM (NH4)2SO4, 50mM DTT, RNase Inhibitor 5U / μL): nuclease-free water in a volume ratio of 50:25:5:100:70.

[0047] The amplification reaction program is shown in Table 3: Table 3

[0048] Note: * For high concentration samples (>=5.00E+05 copies / mL), the number of amplification cycles can be reduced by 6.

[0049] After a round of specific RT-PCR amplification, PooL1 amplification products, i.e., DNA fragments of the protease region and part of the reverse transcriptase region, and PooL2 amplification products, i.e., DNA fragments of part of the reverse transcriptase region and the integrase region, can be obtained. After the PooL1 amplification products and the PooL2 amplification products are mixed, the entire drug resistance detection area including protease, reverse transcriptase and integrase is obtained.

[0050] (3) Purification of RT-PCR amplification products by magnetic beads: Purify the RT-PCR product using commercially available DNA purification magnetic beads, such as AMPure XP magnetic beads, SuoShi Bio DNA purification magnetic beads (Cat. No.: JNC10001); wherein the volume ratio of magnetic beads to RT-PCR reaction product is 0.8:1 (AMPure XP magnetic beads are used in this example); Specific purification process: Remove the DNA purification magnetic beads 30 minutes in advance, vortex thoroughly, and let stand at room temperature. Mix the PooL1 amplification product and the PooL2 amplification product and centrifuge them to the bottom of the tube (the instant centrifugation means: centrifuge for 1-5 seconds at 400-1200rpm). Pipette 80 μL of DNA purification magnetic beads into the PCR product, vortex (you can also use a pipette to gently blow 10 times) to mix thoroughly, and incubate at room temperature for 3-5 minutes. Centrifuge the PCR tube instantaneously and place it on a magnetic stand to separate the magnetic beads and liquid, and wait for the solution to clarify (about 3-5 minutes). Carefully remove the supernatant with a pipette. Add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat step f) for a total of two rinses. Centrifuge the PCR tube instantaneously, put it back on the magnetic stand, and use a 10μL pipette to remove the ethanol remaining at the bottom. Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until the surface of the beads is matte, and be careful not to over-dry the beads. Remove the PCR tube from the magnetic rack, add enzyme-free water for elution (about 22~100μL), vortex (you can also use a pipette to gently blow 10 times) to mix thoroughly, and leave at room temperature for 2 minutes. Centrifuge the PCR tube instantaneously and place it in the magnetic rack to stand. After the solution is clarified, carefully transfer the supernatant to a new EP tube, and do not touch the magnetic beads. This supernatant liquid is the purified target DNA fragment product. (4) Quality inspection of the target fragment product; Concentration determination: Use Qubit 3.0 or 4.0 and Qubit 1X dsDNA HS Kit to determine the concentration of the amplified product; Fragment length analysis: Bioptic Qsep 100 fully automated nucleic acid and protein analysis system can also be used to measure DNA fragment length using other equivalent instruments. The length of the amplified product mixture of PooL1 and PooL2 is about 1550 bp.

[0051] Example 2 This example is used to illustrate the construction of NGS sequencing library The library was constructed using a magnetic bead-coupled transposase library construction kit. Since the transposase adds a universal adapter sequence to the fragmented DNA while fragmenting the DNA, the DNA fragmentation reaction can be used as a template for the downstream index amplification process to form a complete library. At the same time, the transposase is coupled to the magnetic beads, and the library can be constructed without homogenizing the DNA as long as the input amount is within a certain range.

[0052] (1) DNA fragmentation and linker addition: A certain volume (the input amount is in the range of 0.1-100 ng) of the purified product of the amplification product in Example 1 was taken according to the concentration conversion to construct a library.

[0053] Thaw the reagents on ice, mix well, centrifuge briefly to collect the bottom of the tube, and place on ice for later use; Place the PCR tubes on ice and configure the following system as shown in Table 4: Table 4

[0054] Wherein, X: adjusted according to the template concentration, the input volume does not exceed 14 μL, and the total amount is in the range of 0.1~100 ng.

[0055] Use a pipette to gently pipette 20 times to mix thoroughly. Place the reaction tube in a PCR instrument and run the following reaction program, see Table 5: Table 5

[0056] (2) Termination of fragmentation reaction: After the fragmentation reaction is completed, quickly remove the sample from the PCR instrument, add 5 μL Stop buffer (50 mM EDTA, pH 8.0), gently pipette to mix, then put it back into the PCR instrument and incubate at 55°C for 5 min before taking it out.

[0057] (3) Index PCR amplification: Following the fragmentation product from the previous step, set up the following reaction on ice, see Table 6: Table 6

[0058] Among them: N5XX+N7XX index primers (usually containing multiple primer pairs with different sequences) are used to adapt to the Illumina sequencing platform and distinguish the index sequences of different samples.

[0059] The general sequence of N5XX is: 5′-AATGATACGGCGACCACCGAGATCTACAC[NNNNNNNN]TCGTCGGCAGCGTC-3′ (SEQ IDNO.5) The general sequence for N7XX is: 5′-CAAGCAGAAGACGGCATACGAGAT[NNNNNNNN]GTCTCGTGGGCTCGG-3′ (SEQ ID NO. 6).

[0060] Wherein N is any base of A, T, C, or G.

[0061] Use a pipette to mix gently or cover the tube and vortex to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0062] Different labels can be used for different samples, and dozens to hundreds of samples can be sequenced at one time, greatly improving detection throughput and reducing costs.

[0063] Place the PCR tube in a PCR instrument and perform the following reactions, as shown in Table 7: Table 7

[0064] Note: *When the DNA input amount is between 0.1 and 2 ng, the amplification cycle is 13 to 14 cycles; when the DNA input amount is between 2 and 10 ng, the amplification cycle is 11 to 12 cycles; when the DNA input amount is between 10 and 100 ng, the amplification cycle is 9 to 10 cycles.

[0065] (4) Library magnetic bead purification Remove the DNA purification beads 30 minutes in advance, vortex thoroughly, and let stand at room temperature. Pipette 44 μL of DNA purification beads (volume ratio of beads to library PCR product = 0.8:1) into the PCR product, vortex or use a pipette to gently blow 10 times to mix thoroughly, and incubate at room temperature for 5 minutes. Centrifuge the PCR tube instantly and place it on a magnetic stand to separate the beads and liquid. Wait for the solution to become clear (about 3 to 5 minutes). Carefully remove the supernatant with a pipette. Add 200 μl of freshly prepared 80% ethanol to rinse the beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat step e for a total of two rinses. Centrifuge the PCR tube instantly, put it back on the magnetic stand, and use a 10μL pipette to remove the ethanol remaining at the bottom. Keep the PCR tube in the magnetic stand at all times, open the lid and air-dry the beads until the surface of the beads is matte. Be careful not to over-dry the beads. Remove the PCR tube from the magnetic rack, add 22 μL enzyme-free water for elution, vortex or use a pipette to gently blow to mix thoroughly, and leave at room temperature for 2 minutes. Centrifuge the PCR tube instantaneously and place it on the magnetic rack to stand. After the solution is clear, carefully transfer 20 μL of the supernatant to a new EP tube without touching the magnetic beads. This supernatant liquid is the library product. The library product can be stored at 4°C for about a week and at -20°C for a month. (5) Library quality inspection: Library concentration determination: Use the Qubit 3.0 or 4.0 instrument and the matching Qubit 1X dsDNA HS Kit to determine the concentration of the amplified product; Library length analysis: Bioptic Qsep 100 fully automatic nucleic acid protein analysis system or other equivalent instruments are used to measure the length of DNA fragments. The main peak is 450-600 bp, and the average fragment is about 450 bp.

[0066] (6) Library sequencing: Illumina sequencing platform, using PE150 sequencing mode, 0.1Gb / sample data volume on the machine.

[0067] (7) Bioinformatics analysis: Compare and analyze the data and output a report on drug resistance results.

[0068] Example 3: Test results of 4 samples tested by the combination test of the present invention According to the schemes described in Example 1 and Example 2, 4 HIV-1 samples (referred to as samples 1-4) were tested.

[0069] The results of PCR amplification product fragment analysis of samples 1-4 are as follows Figure 2-5 ;in, Figure 2 This is the fragment analysis diagram of the PCR amplification product of sample 1; Figure 3 This is the fragment analysis diagram of the PCR amplification product of sample 2; Figure 4 This is the fragment analysis diagram of the PCR amplification product of sample 3; Figure 5 This is the PCR amplification product fragment analysis diagram of sample 4; it can be seen that the sample amplification fragment has high specificity and the length consistency of the amplification fragments of different samples is good.

[0070] Sample 1-4 library fragment analysis diagram Figure 6-9 ,in, Figure 6 This is the library fragment analysis diagram of sample 1; Figure 7 This is the library fragment analysis diagram of sample 2; Figure 8 This is the library fragment analysis diagram of sample 3; Fig. 9 This is the analysis diagram of the library fragments of sample 4. The results show that the library fragments are concentrated and the peaks between different samples are consistent. Table 8 is the quality control statistics of the sequencing data of samples 1-4. It can be seen that there are many matching reads in the offline data, high effective data utilization, high sequencing depth, and high coverage of the detection area.

[0071] Table 8 Sequencing data quality control statistics of samples 1-4

[0072] Table 9, statistics of drug-resistant gene mutation information of sample 3, for the protease region (PR), reverse transcriptase region (RT) and integrase region (IN), the site mutation information of different mutation frequencies ranging from 11% to more than 99.9% can be accurately obtained.

[0073] Table 9 Statistics of drug resistance gene mutation information of sample 3

[0074] Table 10 Statistical report on drug resistance information of samples 1-4

[0075] Note: sensitive: 0~9; potential low-level resistance: 10~14; low-level resistance: 15~29; moderate resistance: 30~59; high resistance: >= 60 Table 10, statistical report of drug resistance information of 4 example samples, including annotation information of drug-resistant variant amino acids, and sensitivity and mutation resistance scores of 25 common antiviral drugs including protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and integrase inhibitors (INSTI) after database evaluation, which is convenient for doctors to guide patients' medication according to the report information, increase patients' survival time and save medication costs.

[0076] By using the scheme provided by the present invention, the HIV-1 RNA POL gene protease, reverse transcriptase and integrase drug resistance detection region can be amplified and enriched in one step, and combined with high-throughput deep sequencing technology, the drug resistance mutation can be detected quickly, and the inferior drug-resistant strains with an abundance of less than 20% can be well detected. At the same time, the entire POL gene drug resistance detection region is covered, and a complete sequence is assembled to provide effective information for virus typing and epidemiological research. The method has the advantages of simple operation, short time consumption, high sensitivity, strong specificity, covering the HIV-1 POL gene protease, reverse transcriptase and integrase region, the whole drug resistance detection region, low cross-contamination risk, low cost, high throughput, etc., and is suitable for application and promotion.

[0077] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for detecting HIV-1 drug resistance genes based on NGS, characterized in that: The steps include: S1. Extract RNA from HIV-1 serum or plasma samples; S2, amplification and enrichment of drug resistance detection regions: one-step RT-PCR amplification of the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene, and detection of the amplified products; S3, library construction: fragmentation, linker addition, and tagging of the S2 amplified product for simultaneous enrichment to construct a sequencing library; S4, sequencing on the machine; S5. Data analysis: Based on the sequencing data in step S4, the genotypes of the protease, reverse transcriptase, and integrase regions of the HIV-1 POL gene and the drug resistance assessment are obtained.

2. The detection method according to claim 1, characterized in that: In step S2, PooL1 is amplified by primer pair PooL1_F_1 and PooL1_R_1; PooL2 is amplified by primer pair PooL2_F_1 and PooL2_R_1; PooL1 and PooL2 constitute the HIV-1 drug resistance gene region including the protease region, reverse transcriptase region and integrase region of the POL gene; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2; The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.

4.

3. The detection method according to claim 1, characterized in that: In step S2, the RT-PCR reaction system includes: reverse transcriptase, DNA polymerase, dNTPs, buffer and amplification primers.

4. The detection method according to claim 1, characterized in that: The RT-PCR reaction procedure includes the following: Reverse transcription: temperature 42℃~55℃, time 5~40 minutes, 1 cycle; cDNA pre-denaturation: temperature 94℃~98℃, time 10~300 seconds, 1 cycle; Denaturation: temperature 94℃~98℃, time 10~60 seconds, annealing, temperature 56~66℃, time 20~60 seconds, extension, temperature 72℃, time 30~300 seconds, 35~50 cycles; Final extension, 68℃~72℃, time is 2~20 minutes, 1 cycle.

5. The detection method according to claim 1, characterized in that: In S3, the steps of fragmentation, linker addition and tagging simultaneous enrichment operations include: 1) DNA fragmentation and adapter addition: Prepare a reaction system containing 5× reaction buffer, tagment beads, DNA and ddH2O, and react at a specific temperature and time to fragment the DNA and add adapters; 2) Terminate the fragmentation reaction; 3) Index PCR amplification: Using the fragmented product as a template, prepare a reaction system containing the fragmented product, Ampli Mix, and N5XX + N7XX index primers, perform PCR amplification, and obtain the corresponding library.

6. The detection method according to claim 1, characterized in that: Step S2 also includes a step of purifying the amplified product; the purification is performed using magnetic beads, and the ratio of the magnetic beads is 0.5X to 2.0X.

7. A specific primer combination for one-step RT-PCR amplification of the protease region, reverse transcriptase region and integrase region of HIV-1 POL gene, characterized in that: These include: Primer pair PooL1_F_1 and PooL1_R_1 for amplification of PooL1, and primer pair PooL2_F_1 and PooL2_R_1 for amplification of PooL2; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4; The PooL1 and PooL2 constitute the HIV-1 drug resistance gene region including the POL gene protease region, reverse transcriptase region and integrase region.

8. A kit comprising the primer combination according to claim 7.

9. Use of the detection method according to any one of claims 1 to 6, or the primer combination according to claim 7, or the kit according to claim 8 in HIV-1 drug resistance monitoring, prognosis assessment, epidemiological research or drug development.

10. A detection system for HIV-1 drug resistance genes based on NGS, characterized in that: include: A sample processing module for extracting RNA from HIV-1 serum or plasma samples; an amplification module for one-step RT-PCR amplification of the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene; The library construction module is used to perform fragmentation, adapter addition, and tagging of the amplified products for simultaneous enrichment operations; A sequencing module is used to perform sequencing on the constructed sequencing library; The data analysis module is used to analyze sequencing data to obtain the genotype and drug resistance evaluation of the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene.

11. The detection system according to claim 10, characterized in that: The amplification module uses a specific primer combination to amplify the protease region, reverse transcriptase region and integrase region of the HIV-1 POL gene; the specific primer combination includes: Primer pair PooL1_F_1 and PooL1_R_1 for amplification of PooL1, and primer pair PooL2_F_1 and PooL2_R_1 for amplification of PooL2; The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1; The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2 The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3; The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4; The PooL1 and PooL2 constitute the HIV-1 drug resistance gene region including the POL gene protease region, reverse transcriptase region and integrase region.

12. The detection system according to claim 10, characterized in that: The library construction module uses the transposase method to perform fragmentation, linker addition and tagging operations.

13. The detection system according to claim 10, characterized in that: The data analysis module obtains the genotype and drug resistance evaluation of the protease, reverse transcriptase and integrase regions of the HIV-1 POL gene through bioinformatics analysis or online database analysis.

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