A detection method, kit and application of NGS-based HIV-1 drug resistance genes

Abstract

The present invention belongs to the field of detection technology, and specifically discloses a detection method, kit and application of HIV-1 drug resistance genes based on NGS. The detection method includes the steps of: S1, extracting RNA from an HIV-1 serum or plasma sample; S2, amplifying and enriching the drug resistance detection region: performing one-step RT-PCR to amplify the protease region, reverse transcriptase region and integrase region of the HIV-1 POL gene, and detecting the amplification product; S3, library construction: performing fragmentation, adapter addition and tag addition and synchronous enrichment operations on the amplification product in S2 to construct a sequencing library; S4, performing on-machine sequencing; S5, data analysis: according to the sequencing data in step S4, analyzing and obtaining the genotypes and drug resistance evaluations of the protease and reverse transcriptase regions and integrase regions of the HIV-1 POL gene. The present invention only requires one-step RT-PCR, and does not require two-round nested PCR to specifically enrich the drug resistance detection region of the HIV-1 POL gene.

Description

Technical Field

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

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

[0003] HIV is divided into HIV-1 and HIV-2 types. HIV-1 is prevalent globally and can be divided into 4 groups: M, N, O and P. Among them, the most important group M can be divided into subtypes A-D, F-H, J and K. The strains of groups N, O and P are mainly in Central Africa with a relatively low prevalence. HIV-2 is mainly in the African region and has more than 7 subtypes A-G. The HIV genome is single-stranded RNA, about 9,000 bases, containing 9 genes, encoding a total of 15 proteins. Among them, the polymerase (pol) gene encodes a variety of viral enzymes, including protease, reverse transcriptase and integrase, etc.

[0004] Antiretroviral therapy (ART), as an effective means to reduce the death of HIV-infected individuals and reduce the transmission of HIV, is prone to gene mutations and drug resistance due to the high-speed replication of the AIDS virus itself and the lack of self-proofreading function during the replication process. At the same time, under the selective pressure of long-term antiviral therapy drugs, the probability of drug-resistant mutations of the AIDS virus increases greatly. The occurrence and development of drug-resistant mutations will affect the efficacy of antiviral therapy. 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 by RT-PCR, and then Sanger sequencing is performed to obtain sequence information, which is compared with the reference nucleic acid sequence to evaluate whether there are mutations at the drug resistance sites. The Sanger sequencing method has some technical disadvantages, mainly low sensitivity, poor detection effect for variations with an abundance below 20% or even higher, and subjective result interpretation by humans.

[0006] In view of the deficiencies of Sanger sequencing, a method for detecting drug-resistant mutations using next-generation sequencing (NGS) based on deep sequencing has solved these deficiencies and can even detect mutations below 1%. However, the existing methods for detecting drug resistance in the HIV-1 RNA POL gene have the following deficiencies:

[0007] 1. The detection region coverage is incomplete, and the protease, reverse transcriptase, and integrase regions of the HIV-1 RNA POL gene are not covered simultaneously, which cannot meet the requirements of the detection regions recommended by the latest guidelines;

[0008] 2. When capturing and enriching the drug-resistant regions of the HIV-1 RNA POL gene, two rounds of PCR are involved. One type is the method of first performing RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and then performing nested PCR in the second round for amplification and enrichment. Another type is the method of first synthesizing cDNA by reverse transcription and then performing one round of PCR and nested PCR in the second round for amplification and enrichment. The enriched products are detected and analyzed by first-generation sequencing or next-generation sequencing. It is impossible to achieve highly sensitive and highly specific capture and enrichment of protease, reverse transcriptase, and integrase drug-resistant detection regions with one round of RT-PCR. There are problems such as cumbersome operation, long time consumption, and the risk of aerosol cross-contamination during the operation of opening the lid and transferring tubes. Summary of the Invention

[0009] In view of the above deficiencies, the present invention provides a detection method, kit, and application for detecting HIV-1 drug-resistant genes based on NGS one-step RT-PCR. Through specific primers, one-step RT-PCR is used to amplify and enrich the protease, reverse transcriptase region, and integrase region of the HIV-1 POL gene. Combined with the construction of a next-generation sequencing library using the transposase method, sequencing on a machine, and bioinformatics analysis or online database analysis, the genotypes of the protease, reverse transcriptase region, and integrase region of the HIV-1 POL gene can be quickly obtained, and the HIV-1 drug resistance can be evaluated.

[0010] The technical solution adopted by the present invention is as follows:

[0011] <First aspect>

[0012] A detection method for HIV-1 drug-resistant genes based on NGS, comprising the following steps:

[0013] S1. Extract the RNA of the HIV-1 serum or plasma sample;

[0014] S2. Amplification and enrichment of the drug resistance detection region: Use one-step RT-PCR to amplify the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene, and detect the amplification products.

[0015] S3. Library construction: Perform fragmentation, adapter ligation, and tag addition and synchronous enrichment operations on the amplification products in S2 to construct a sequencing library.

[0016] S4. Sequencing on the machine.

[0017] S5. Data analysis: According to the sequencing data in step S4, analyze to obtain the genotypes of the protease and reverse transcriptase regions, and integrase region of the HIV-1 POL gene and drug resistance assessment.

[0018] 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; the Pool1 (protease and partial reverse transcriptase region (reference sequence OQ747186.1: 2074-3632)) and Pool2 (partial 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.

[0019] The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1;

[0020] The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2

[0021] The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3;

[0022] The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4.

[0023] Generally, immediately extract the serum sample after separation or store it at -80°C; the plasma sample sampling requires fresh EDTA anticoagulated whole blood. It is recommended to separate the plasma within 2 hours, not exceeding 6 hours; the concentration of the HIV-1 RNA virus sample is not less than 500 copies / mL, extract >=200 μL of the plasma sample, and the total RNA volume is about 50 μL. High-concentration samples can be diluted before use, and avoid repeated freezing and thawing.

[0024] In step S2, the specific primer design process involves reference sequence selection, sequence analysis and processing, and primer design process: Download all HIV-1 complete genome sequences from the NCBI database, retain genome sequences with a length not less than 98% of the reference genome sequence NC_001802.1, filter low-quality sequences (including those with more than 10 uncertain bases N), remove duplicate sequences with a 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) processing on the remaining representative reference sequences, identify conserved sequences from multiple related sequences, select the most representative reference sequence in the target region, and then use primer3 or other primer design software to design amplification primers for the target region sequence of the most representative reference sequence. After the amplification primers are designed, check the coverage of the primer sequences with the representative reference sequences, and appropriately add some degenerate bases to improve the inclusiveness of the primer sequences for different reference sequences.

[0025] In step S2, the RT-PCR reaction system includes: reverse transcriptase, DNA polymerase, dNTPs, buffer, and amplification primers.

[0026] Mix high-concentration reverse transcriptase, DNA polymerase, dNTPs, and 5X buffer, and supplement appropriate nuclease-free water to obtain 2X One Step RT-PCR master mix; the concentrations of each component in 2X One Step RT-PCR master mix are dNTPs 5 mM, reverse transcriptase 5 U / μL, DNA polymerase 0.08 U / μL, and 2X buffer.

[0027] The reverse transcriptase includes: commercially available common MMLV reverse transcriptase; preferably RNase H- MMLV reverse transcriptase.

[0028] The 5X buffer is: containing 125 mM Tris-HCl (pH value 8.3), 250 mM KCl, 15 mM MgCl2, 50 mM (NH4)2SO4, 50 mM DTT, and RNase Inhibitor 5 U / μL.

[0029] The DNA polymerase is: commercially available common DNA polymerase, including but not limited to Taq DNA polymerase, PfuDNA polymerase, Q5® High-Fidelity DNA Polymerase, etc., and ordinary Taq DNA polymerase can achieve the effect.

[0030] The amplification primers include: primer pair PooL1_F_1 and PooL1_R_1 for amplifying PooL1; and primer pair PooL2_F_1 and PooL2_R_1 for amplifying PooL2;

[0031] The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1;

[0032] The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2

[0033] The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3;

[0034] The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4;

[0035] The final reaction concentrations of PooL1_F_1 and PooL1_R_1 are 0.1 - 1 μM, preferably 0.4 μM.

[0036] The final reaction concentrations of PooL2_F_1 and PooL2_R_1 are 0.1 - 1 μM, preferably 0.4 μM.

[0037] The RT-PCR reaction procedure is as follows:

[0038] Reverse transcription: temperature is 42°C - 55°C, time is 5 - 40 minutes, 1 cycle;

[0039] cDNA pre-denaturation: temperature is 94°C - 98°C, time is 10 - 300 seconds, 1 cycle;

[0040] Denaturation, temperature is 94°C - 98°C, time is 10 - 60 seconds, annealing, temperature is 56 - 66°C, time is 20 - 60 seconds, extension, temperature is 72°C, time is 30 - 300 seconds, 35 - 50 cycles;

[0041] Final extension, 68°C - 72°C, time is 2 - 20 minutes, 1 cycle.

[0042] Furthermore, the RT-PCR reaction procedure is as follows:

[0043] Reverse transcription: temperature is 50°C, time is 30 minutes, 1 cycle;

[0044] cDNA pre-denaturation: temperature is 95°C, time is 180 seconds, 1 cycle;

[0045] Denaturation, temperature is 95°C, time is 20 seconds, annealing, temperature is 60°C, time is 45 seconds, extension, temperature is 72°C, time is 120 seconds, 42 - 48 cycles.

[0046] Final extension: 72°C, 5 minutes, 1 cycle.

[0047] Step S2 also includes a step of purifying the amplified product.

[0048] The purification is carried out by magnetic bead purification, and the ratio of magnetic beads is 0.5X~2.0X, preferably 0.8X.

[0049] Step S3: constructing a sequencing library includes the construction methods of Illumina, MGI library, or Ion Torrent library.

[0050] In step S3, the DNA sample input amount is 0.1ng~100ng. Tagment-Beads (transposase coupled to magnetic beads) is preferably used for fragmentation and adapter addition. The fragmented product is added to the Stop buffer to terminate the reaction. Then, the amplification enzyme system and N5XX and N7XX tag primers are added for amplification. Amplification is performed for 9~14 cycles. The product is purified using magnetic beads. After quality inspection and pooling, the purified product becomes the library to be sequenced.

[0051] In S3, the steps of fragmentation, adapter addition, and tagging simultaneous enrichment include:

[0052] 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. Incubate at a specific temperature and time to fragment the DNA and add adapters.

[0053] 2) Stop the fragmentation reaction: After the reaction is complete, remove the sample, add Stop buffer, mix well, and return it to the PCR instrument for incubation before removing it;

[0054] 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 amplify the mixture using PCR. Finally, perform the reaction at a specific temperature and time. Purify the reaction product to obtain the corresponding library.

[0055] The target detection region is fragmented, adapters are added, tags are added and enriched simultaneously to construct a sequencing library. Any library construction method can be selected. After quality inspection, different libraries are pooled and mixed, then diluted and denatured to the concentration required for sequencing.

[0056] The primers for PooL1 are primer set PooL1_1, and the primers for PooL2 are primer set PooL2_1, see the table below:

[0057]

[0058] Among them, R and Y are degenerate bases.

[0059] <Second aspect>

[0060] 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 the HIV-1 POL gene, including the following:

[0061] The primer pair PooL1_F_1 and PooL1_R_1 for amplifying PooL1, and the primer pair PooL2_F_1 and PooL2_R_1 for amplifying PooL2;

[0062] The nucleotide sequence of the PooL1_F_1 is as shown in SEQ ID NO.1;

[0063] The nucleotide sequence of the PooL1_R_1 is as shown in SEQ ID NO.2

[0064] The nucleotide sequence of the PooL2_F_1 is as shown in SEQ ID NO.3;

[0065] The nucleotide sequence of the PooL2_R_1 is as shown in SEQ ID NO.4;

[0066] The 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.

[0067] <Third aspect>

[0068] The present invention also provides a kit including the primer combination as described above.

[0069] <Fourth aspect>

[0070] The present invention also provides an application of the detection method, primer combination, or kit as described above in HIV-1 drug resistance monitoring, prognosis assessment, epidemiological research or drug development.

[0071] <Fifth aspect>

[0072] The present invention also provides an NGS-based detection system for HIV-1 drug resistance genes, which is characterized by including:

[0073] A sample processing module for extracting RNA from HIV-1 serum or plasma samples;

[0074] 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;

[0075] A library construction module for fragmenting, adapter-ligating, and tag-adding and synchronously enriching the amplification products;

[0076] A sequencing module for performing on-machine sequencing on the constructed sequencing library;

[0077] A data analysis module for analyzing the sequencing data to obtain the genotypes and drug resistance evaluations of the protease and reverse transcriptase regions and the integrase region of the HIV-1 POL gene.

[0078] In the amplification module, a specific primer combination is used to amplify the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene; the specific primer combination includes:

[0079] The primer pair PooL1_F_1 and PooL1_R_1 for amplifying PooL1, and the primer pair PooL2_F_1 and PooL2_R_1 for amplifying PooL2;

[0080] The nucleotide sequence of PooL1_F_1 is as shown in SEQ ID NO.1;

[0081] The nucleotide sequence of PooL1_R_1 is as shown in SEQ ID NO.2

[0082] The nucleotide sequence of PooL2_F_1 is as shown in SEQ ID NO.3;

[0083] The nucleotide sequence of PooL2_R_1 is as shown in SEQ ID NO.4;

[0084] 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.

[0085] The library construction module uses the transposase method for fragmenting, adapter-ligating, and tag-adding operations.

[0086] The data analysis module obtains the genotypes and drug resistance evaluations of the protease and reverse transcriptase regions and the integrase region of the HIV-1 POL gene through bioinformatics analysis or online database analysis.

[0087] Compared with the prior art, the present invention has the following beneficial effects:

[0088] 1. Only one-step RT-PCR is required, and two-round nested PCR is not needed, which can highly sensitively and specifically enrich the drug resistance detection region of the HIV-1 POL gene;

[0089] 2. The library construction is carried out by the method of magnetic bead coupling transposase, without the need for normalization. The DNA input amount can directly perform downstream library construction from 0.1 to 100 ng, with a wide compatibility range.

[0090] 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

[0091] By reading the following detailed description of the non - restrictive embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more apparent:

[0092] Figure 1 It is a flow chart of the detection method for HIV - 1 drug - resistant genes based on NGS of the present invention;

[0093] Figure 2 It is the analysis result of the PCR amplification product fragments of sample 1 in Example 3;

[0094] Figure 3 It is the analysis result of the PCR amplification product fragments of sample 2 in Example 3;

[0095] Figure 4 It is the analysis result of the PCR amplification product fragments of sample 3 in Example 3;

[0096] Figure 5 It is the analysis result of the PCR amplification product fragments of sample 4 in Example 3;

[0097] Figure 6 It is the analysis diagram of the library fragments of sample 1 in Example 3;

[0098] Figure 7 It is the analysis diagram of the library fragments of sample 2 in Example 3;

[0099] Figure 8 It is the analysis diagram of the library fragments of sample 3 in Example 3;

[0100] Figure 9 It is the analysis diagram of the library fragments of sample 4 in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0101] The present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can still be made. These all belong to the protection scope of the present invention.

[0102] Example 1

[0103] Figure 1Flowchart of the method for detecting HIV-1 drug resistance genes based on NGS of the present invention; this example is used to illustrate the acquisition of target fragments in the protease, reverse transcriptase region, and integrase region of the HIV-1 POL gene

[0104] 1. Sample extraction: Extract the HIV-1 RNA sample from the serum or plasma sample of AIDS patients (serum sample is used in this example). First, centrifuge the plasma or serum at 1600 - 2000 xg (2000 xg is used in this example) at 4°C for 10 - 20 minutes (15 minutes of centrifugation in this example), and immediately extract it or store it at -80°C. Then use QIAGEN Viral RNA Mini Kit (product number: 52904 / 52906) or Biosino Bio-technology & Science Inc. viral extraction kit (magnetic bead method) (product number: SDK60104), and perform according to the specific requirements of the instructions.

[0105] 2. Specific RT-PCR amplification: Use the HIV-1 specific RT-PCR primer pair to amplify the RNA extracted in step (1). The primer set is shown in Table 1:

[0106] Table 1

[0107]

[0108] Among them, R and Y are degenerate bases.

[0109] The reaction system of one-step RT-PCR amplification is shown in Table 2:

[0110] Table 2

[0111]

[0112] Among them, the amplification primers in Pool1 are Pool1_F_1 and Pool1_R_1, the working solution concentration of the primers is 10 μM, and the final concentration is 0.4 μM; the amplification primers in Pool2 are Pool2_F_1 and Pool2_R_1, the working solution concentration of the primers is 10 μM, and the final concentration is 0.4 μM.

[0113] Among them, the concentrations of each component of 2X One Step RT-PCR master mix: dNTPs is 5 mM, RNase H- MMLV reverse transcriptase is 5 U / μL, Taq DNA polymerase is 0.08 U / μL, 2X buffer.

[0114] Among them, the 2X One Step RT-PCR master mix is obtained by uniformly mixing 100 mM dNTPs: 50 U / μL MMLV reverse transcriptase without RNase H: 4 U / μL Taq DNA polymerase: 5X buffer (containing 125 mM Tris-HCl (pH 8.3), 250 mM KCl, 15 mM MgCl2, 50 mM (NH4)2SO4, 50 mM DTT, 5 U / μL RNase Inhibitor): nuclease-free water in a volume ratio of 50:25:5:100:70.

[0115] The amplification reaction program is shown in Table 3:

[0116] Table 3

[0117]

[0118] Remark: *For high-concentration samples (>=5.00E+05 copies / mL), 6 amplification cycles can be reduced.

[0119] After one round of specific RT-PCR amplification, the PooL1 amplification product, namely the DNA fragments of the protease region and part of the reverse transcriptase region, and the PooL2 amplification product, namely the DNA fragments of part of the reverse transcriptase region and the integrase region, can be obtained. After mixing the PooL1 amplification product and the PooL2 amplification product, the entire drug resistance detection region including protease, reverse transcriptase, and integrase is obtained.

[0120] (3) Magnetic bead purification of RT-PCR amplification products:

[0121] Commercially available DNA purification magnetic beads are used to purify the RT-PCR products, such as AMPure XP magnetic beads, Biosino Bio-technology & Science Inc. DNA purification magnetic beads (product number: JNC10001); among them, the volume ratio of magnetic beads to the RT-PCR reaction product is 0.8:1 (AMPure XP magnetic beads are used in this example);

[0122] Specific purification process:

[0123] Take out the DNA purification magnetic beads 30 minutes in advance, vortex thoroughly, and let stand at room temperature. Mix the amplification products of PooL1 and PooL2 and centrifuge briefly to the bottom of the tube (the brief centrifugation means: centrifuge at 400 - 1200 rpm for 1 - 5 s). Pipette 80 μL of DNA purification magnetic beads into the PCR product, vortex (or gently pipette 10 times with a pipette) to mix well, and incubate at room temperature for 3 - 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic rack to separate the magnetic beads and the liquid. Wait until the solution is clear (about 3 - 5 minutes). Carefully remove the supernatant with a pipette. Add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat step f), for a total of two washes. Centrifuge the PCR tube briefly, put it back on the magnetic rack, and use a 10 μL pipette to suck out the residual ethanol at the bottom. Keep the PCR tube on the magnetic rack all the time, open the lid and air-dry the magnetic beads until the surface of the magnetic beads has no luster, taking care not to over-dry the magnetic beads. Take the PCR tube out of the magnetic rack, add enzyme-free water for elution (about 22 - 100 μL), vortex (or gently pipette 10 times with a pipette) to mix well, and let stand at room temperature for 2 minutes. Centrifuge the PCR tube briefly and place it on the magnetic rack to stand. After the solution is clear, carefully transfer the supernatant to a new EP tube, taking care not to touch the magnetic beads. This supernatant liquid is the purified target DNA fragment product. (4) Quality inspection of the target fragment product;

[0124] Concentration determination: Use qubit 3.0 or 4.0 and Qubit 1X dsDNA HS Kit to determine the concentration of the amplification product;

[0125] Fragment length analysis: The Bioptic Qsep 100 fully automatic nucleic acid and protein analysis system or other equivalent instruments can also be used to determine the length of the DNA fragment. The mixture of the amplification products of PooL1 and PooL2 is about 1550 bp.

[0126] Example 2 This example is used to illustrate the construction of the NGS sequencing library

[0127] Use a magnetic bead-coupled transposase method library construction kit to construct the library. Since the transposase adds a universal adapter sequence to the fragmented DNA while fragmenting the DNA, after the DNA fragmentation reaction terminates, it can be used as a template for the downstream index addition and amplification process to form a complete library. At the same time, the transposase is coupled with the magnetic beads, and within a certain range of input amounts, DNA does not need to be normalized to construct the library.

[0128] (1)DNA fragmentation and adapter addition:

[0129] For the purified product of the amplification product in Example 1, take a certain volume according to the concentration conversion (the input amount is in the range of 0.1 - 100 ng) to construct the library.

[0130] Thaw the reagent on ice. After mixing evenly, centrifuge instantaneously to collect the bottom of the tube and place it on ice for standby;

[0131] Place the PCR tube on ice and prepare the following system as shown in Table 4:

[0132] Table 4

[0133]

[0134] Among them, X: Adjust 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.

[0135] Gently pipette 20 times to mix evenly. Place the reaction tube in the PCR instrument and run the following reaction program, as shown in Table 5:

[0136] Table 5

[0137]

[0138] (2) Termination of fragmentation reaction: After the fragmentation reaction is completed, quickly take out the sample from the PCR instrument, add 5 μL of Stop buffer (50 mM EDTA, pH 8.0), gently pipette and mix evenly, then put it back into the PCR instrument again. Take it out after incubating at 55 °C for 5 min.

[0139] (3) Index PCR amplification:

[0140] Based on the above-step fragmentation product, prepare the following reaction on ice, as shown in Table 6:

[0141] Table 6

[0142]

[0143] Among them: N5XX + N7XX index primers (usually including multiple primer pairs with different sequences), which are used to adapt to the illumina sequencing platform and distinguish the tag sequences of different samples.

[0144] The common sequence of N5XX is:

[0145] 5′-AATGATACGGCGACCACCGAGATCTACAC[NNNNNNNN]TCGTCGGCAGCGTC-3′ (SEQ ID NO.5)

[0146] The common sequence of N7XX is:

[0147] 5′-CAAGCAGAAGACGGCATACGAGAT[NNNNNNNN]GTCTCGTGGGCTCGG-3′ (SEQ ID NO.6).

[0148] Wherein N is any one of the bases A, T, C, and G.

[0149] Gently pipette to mix well or cover the tube cap and vortex to mix well, and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0150] Different samples can use different labels. Dozens to hundreds of samples can be loaded onto the machine for one sequencing, greatly improving the detection throughput and significantly reducing the cost.

[0151] Place the PCR tube in a PCR instrument and perform the following reaction, as shown in Table 7:

[0152] Table 7

[0153]

[0154] Note: *When the DNA input amount is between 0.1 and 2 ng, 13 - 14 amplification cycles are selected; when the DNA input amount is between 2 and 10 ng, 11 - 12 amplification cycles are selected; when the DNA input amount is between 10 and 100 ng, 9 - 10 amplification cycles are selected.

[0155] (4) Library magnetic bead purification

[0156] Take out the DNA purification magnetic beads 30 minutes in advance, vortex thoroughly, and let stand at room temperature. Pipette 44 μL of DNA purification magnetic beads (the volume ratio of magnetic beads to library PCR product = 0.8:1) into the PCR product, vortex or gently pipette 10 times to mix well, and incubate at room temperature for 5 minutes. Centrifuge the PCR tube instantaneously and place it in a magnetic rack to separate the magnetic beads and the liquid until the solution becomes clear (about 3 - 5 minutes). Carefully remove the supernatant with a pipette. Add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat step e for a total of two washes. Centrifuge the PCR tube instantaneously, place it back on the magnetic rack, and use a 10 μL pipette to suck off the residual ethanol at the bottom. Keep the PCR tube always in the magnetic rack, open the lid and air-dry the magnetic beads until the surface of the magnetic beads has no luster, taking care not to over-dry the magnetic beads. Take the PCR tube out of the magnetic rack, add 22 μL of enzyme-free water for elution, vortex or gently pipette to mix well, and let stand at room temperature for 2 minutes. Centrifuge the PCR tube instantaneously and place it in the magnetic rack to stand. After the solution becomes clear, carefully transfer 20 μL of the supernatant to a new EP tube, taking care not to touch the magnetic beads. This supernatant liquid is the library product. The library product can be stored at 4°C for about one week and at -20°C for one month. (5) Library quality inspection:

[0157] Library concentration determination: Use a Qubit 3.0 or 4.0 instrument and the accompanying Qubit 1X dsDNA HS Kit to determine the concentration of the amplified product;

[0158] Library length analysis: Use a Bioptic Qsep 100 fully automatic nucleic acid and protein analysis system or other equivalent instruments to determine the length of DNA fragments. The main peak is at 450 - 600 bp, and the average fragment is about 450 bp.

[0159] (6) Library loading for sequencing: On the Illumina sequencing platform, use the PE150 sequencing mode and load 0.1 Gb / sample of data.

[0160] (7) Bioinformatics analysis: Conduct comparative analysis on the data and output a drug resistance result report.

[0161] Example 3. Detection results of 4 samples tested by the combination of the present invention

[0162] According to the schemes described in Example 1 and Example 2, 4 HIV-1 samples (denoted as samples 1 - 4) were tested.

[0163] The fragment analysis results of the PCR amplification products of samples 1 - 4 are as Figures 2 - 5 ; among them, Figure 2 is the fragment analysis diagram of the PCR amplification product of sample 1; Figure 3 is the fragment analysis diagram of the PCR amplification product of sample 2; Figure 4 is the fragment analysis diagram of the PCR amplification product of sample 3; Figure 5 is the fragment analysis diagram of the PCR amplification product of sample 4; it can be seen that the amplified fragments of the samples have high specificity and good consistency in the length of the amplified fragments of different samples.

[0164] The library fragment analysis diagrams of samples 1 - 4 are as Figures 6 - 9 ; among them, Figure 6 is the library fragment analysis diagram of sample 1; Figure 7 is the library fragment analysis diagram of sample ②; Figure 8 is the library fragment analysis diagram of sample 3; Figure 9 is the library fragment analysis diagram of sample 4. The results show that the library fragment distribution is concentrated and the peak shapes are consistent among different samples; 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 downloaded data, the utilization rate of valid data is high, the sequencing depth is high, and the coverage of the detection region is high.

[0165] Table 8 Quality control statistics of the sequencing data of samples 1 - 4

[0166]

[0167] Table 9. Statistical information on the variation of drug-resistant genes in the samples of Example 3. For the protease region (PR), reverse transcriptase region (RT), and integrase region (IN), the variation information of sites with different variation frequencies from 11% to over 99.9% can be accurately obtained.

[0168] Table 9 Statistical Information on the Variation of Drug-Resistant Genes in Sample 3

[0169]

[0170] Table 10 Statistical Report on the Drug Resistance Information of Samples 1 - 4

[0171]

[0172] Note: Sensitive: 0 - 9; Potentially low-level drug resistance: 10 - 14; Low-level drug resistance: 15 - 29; Moderate drug resistance: 30 - 59; High-level drug resistance: >= 60

[0173] Table 10 Statistical Report on the Drug Resistance Information of 4 Example Samples, including the annotation information of drug-resistant mutant amino acids, and the sensitivity and mutant drug resistance scores of 25 common antiviral drugs against 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, increasing the patients' survival period and saving medication costs.

[0174] Using the solution provided by the present invention, the protease, reverse transcriptase, and integrase drug resistance detection regions of the HIV-1 RNA POL gene can be amplified and enriched in one step. Combining with high-throughput deep sequencing technology, the detection of drug-resistant mutations can be quickly achieved, and the disadvantaged drug-resistant strains with an abundance of less than 20% can be well detected. At the same time, covering the entire POL gene drug resistance detection region, a complete sequence can be assembled to provide effective information for virus typing and epidemiological research. This method has the advantages of simple operation, short time consumption, high sensitivity, strong specificity, covering the protease, reverse transcriptase, and integrase regions of the HIV-1 POL gene, complete drug resistance detection region, low risk of cross-contamination, low cost, and high throughput, and is suitable for application and promotion.

[0175] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A specific primer combination for one-step RT-PCR amplification of the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene, characterized in that, It includes the following: Primer pairs PooL1_F_1 and PooL1_R_1 for amplifying PooL1, and primer pairs 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; 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.

2. A kit comprising the primer combination according to claim 1.

3. An NGS-based detection system for HIV-1 drug resistance genes, characterized in that, It includes: A sample processing module for extracting RNA from an HIV-1 serum or plasma sample; 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; A library construction module for performing fragmentation, adapter addition, and tag addition and synchronous enrichment operations on the amplification product; A sequencing module for performing on-machine sequencing on the constructed sequencing library; A data analysis module for analyzing sequencing data to obtain the genotypes and drug resistance assessment of the protease and reverse transcriptase regions and integrase region of the HIV-1 POL gene; In the amplification module, a specific primer combination is used to amplify the protease region, reverse transcriptase region, and integrase region of the HIV-1 POL gene; the specific primer combination includes: Primer pairs PooL1_F_1 and PooL1_R_1 for amplifying PooL1, and primer pairs 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; 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.

4. The detection system according to claim 3, wherein The library construction module uses the transposase method for fragmentation, adapter addition, and tag addition operations.

5. The detection system according to claim 3, wherein The data analysis module obtains the genotypes and drug resistance assessment of the protease and reverse transcriptase regions and integrase region of the HIV-1 POL gene through bioinformatics analysis or online database analysis.

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