Primer set and method for detecting drug resistance mutation in HIV-1 RNA integrase region

Abstract

The application discloses a primer group and a method for detecting HIV-1 RNA integrase region drug-resistant mutations. The detection method can be applied to integrase drug-resistant detection of HIV-1 RNA including low-concentration HIV-1 RNA through optimization of a drug-resistant system, an amplification procedure and primer sequences. The detection method can use anticoagulated serum, plasma, semen, urine, cerebrospinal fluid and other human body fluids as samples, adopt a specific primer group, amplify HIV-1 integrase region sequences through a nested PCR method, determine the amplified sequences through a Sanger sequencing method, analyze drug-resistant related gene mutations, judge whether drug resistance exists and the degree of drug resistance by using a genotype drug resistance interpretation system, and thus provide a reference basis for formulating a first-line antiviral treatment, a second-line treatment scheme, a pre / post-exposure prevention drug scheme and a measure for reducing drug resistance.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a primer set for HIV-1 RNA integrase region drug resistance mutations, a method for detecting HIV-1 RNA integrase region drug resistance mutations using the primer set, and a detection kit for HIV-1 RNA integrase region drug resistance mutations containing the primer set. Background Technology

[0002] AIDS (Acquired Immune Deficiency Syndrome) is a syndrome caused by infection with the human immunodeficiency virus (HIV), leading to weakened or deficient immune function, resulting in a series of opportunistic infections and malignant tumors, and in severe cases, death. It is estimated that in 2020, there were approximately 37.7 million people living with HIV globally, with 680,000 deaths, and about 1.5 million new infections each year. Highly active antiretroviral therapy (HAART) can significantly improve the prognosis of people living with HIV, helping to slow viral replication, enhance immunity, and prolong life. It is currently the most effective method for preventing and treating AIDS.

[0003] Currently developed and used antiviral drugs can be divided into six major categories: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INIs), CCR5 receptor inhibitors (RIs), and fusion inhibitors (FIs). In China, the main drugs used to treat AIDS fall into three categories: non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, and protease inhibitors. These three types of drugs can be used alone or in combination to improve treatment efficacy. However, with the research and use of these three types of drugs, HIV-1 drug resistance has been accumulating, weakening the effectiveness of antiviral drugs and thus prompting the development of integrase inhibitor research. INIs are the latest anti-HIV-1 treatment drugs. They mainly target the integrase region, blocking the process of viral genome integration into the human genome and inhibiting other related functions of integrase, thereby rapidly reducing the concentration of HIV-1 RNA in plasma.

[0004] The first integrase inhibitor (INSTI), raltelavin, was launched in the United States in 2007, followed by aviravir and dolutegravir in 2010. Currently, the integrase inhibitors approved and marketed in China include raltelavin, dolutegravir, and aviravir. Bictegravir (trade name: Biktovir) has also been approved for review. Furthermore, some integrase inhibitors are covered by medical insurance. Therefore, resistance testing for integrase drugs is essential.

[0005] Currently, research on integrase resistance in my country is limited. Because INIs have a short market history, a high resistance barrier, and lack integrase in human cells, they have fewer toxic side effects, making them more beneficial for patient treatment. Furthermore, in my country, a viral load (VL) > 1000 copies / mL is generally considered indicative of antiviral therapy failure. Traditional HIV-1 RNA gene resistance detection methods using Sanger sequencing typically only detect HIV-infected individuals with a VL ≥ 1000 copies / mL; when VL < 1000 copies / mL, the viral gene amplification failure rate is high.

[0006] While HAART has improved the survival of people living with HIV since its introduction, 20%-30% of patients still develop low-level viermia (LLV). Compared to patients who have achieved virological suppression, those with low-level viermia have an increased risk of virological failure, poor immune reconstitution, and drug resistance. Because conventional drug resistance testing only guarantees success at viral loads >1000 copies / mL, clinicians generally only test for resistance when the viral load exceeds 1000 copies / mL, but this group accounts for less than 5% clinically. Patients with low viral loads of 50-1000 copies / mL account for approximately 30% clinically, and LLV is associated with HIV-1 drug resistance.

[0007] Therefore, in order to meet market demand, there is an urgent need to develop a method for detecting drug resistance to low concentrations of RNA integrase. Summary of the Invention

[0008] To address the problems of existing technologies, this application provides a method for detecting drug resistance mutations in the HIV-1 RNA integrase region. This method can be applied to the detection of drug resistance at all concentrations of HIV-1 RNA, including low-concentration integrase resistance (below 500 copies / mL for low concentrations and above 500 copies / mL for high concentrations). The method uses human bodily fluids such as serum, plasma, semen, urine, and cerebrospinal fluid as samples. A specific primer set is used to amplify the HIV-1 integrase region using nested PCR. The amplified sequence is then determined using Sanger sequencing. The obtained relevant gene fragment sequences are compared with wild-type sequences and information from drug-resistant strains to analyze drug resistance-related gene mutations. A genotype-based drug resistance interpretation system is used to determine whether drug resistance exists and its degree. This provides a reference for patients in developing first-line antiviral treatment, second-line treatment plans, pre- and post-exposure prophylaxis regimens, and measures to reduce the occurrence of drug resistance.

[0009] In a first aspect, this application provides a primer set for amplifying the HIV-1 RNA integrase region, the primer set comprising:

[0010] The first primer set comprises a first set of upstream primers and a first set of downstream primers, wherein the first set of upstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 1 to 26; and the first set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 27 to 55; and the second primer set comprises a second set of upstream primers and a second set of downstream primers, wherein the second set of upstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 56 to 82; and the second set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 83 to 112.

[0011] Furthermore, in the primer sequence of the primer set, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters R / D / V / Y / N / B represent mixed bases; wherein R represents A+G; D represents A+G+T; V represents A+G+C; Y represents C+T; N represents A+G+C+T; and B represents C+G+T.

[0012] Furthermore, the first set of upstream primers includes four mixed bases: R, D, V, and Y; the first set of downstream primers includes one mixed base: N; the second set of upstream primers includes three mixed bases: R, V, and B; and the second set of downstream primers includes two mixed bases: Y and R.

[0013] Secondly, this application provides a sequencing primer set for sequencing the amplified product of the HIV-1 integrase region gene, wherein the amplified product is obtained by PCR amplification using the primer set described in the first aspect; the sequencing primer set includes at least four primers with nucleotide sequences of SEQ ID NO: 113-138, and the sequencing primer set includes forward sequencing primers and reverse sequencing primers.

[0014] Furthermore, in the sequencing primer sequence, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters R / D represent mixed bases; wherein R represents A+G; and D represents A+G+T.

[0015] Thirdly, this application provides a kit for detecting drug resistance mutations in the integrase region of HIV-1 RNA, the kit comprising RNA extraction reagent, RNA reverse transcription reagent, the primer set described in the first aspect and / or the sequencing primer set described in the second aspect.

[0016] Fourthly, this application provides a method for detecting drug resistance mutations in the HIV-1 RNA integrase region, the detection method comprising:

[0017] Total RNA was extracted from the sample to be tested, and the RNA was reverse transcribed into cDNA.

[0018] Using cDNA as a template, nested PCR amplification was performed using the primer set described in the first aspect to obtain nested PCR amplification products;

[0019] The nested PCR amplification products were analyzed by electrophoresis (e.g., agarose gel electrophoresis).

[0020] The nested PCR amplification products were purified and then sequenced.

[0021] The sequencing results were analyzed to obtain the results of drug resistance mutations in the HIV-1 RNA integrase region.

[0022] Furthermore, the nested PCR amplification includes:

[0023] The first round of PCR amplification uses the aforementioned first primer set as the primers.

[0024] The second round of PCR amplification used the same primers as the second primer set mentioned above.

[0025] Furthermore, the primers used for sequencing the amplified products are the sequencing primer set described in the second aspect.

[0026] Furthermore, the PCR amplification system includes:

[0027] The first round of PCR amplification system, with a reaction volume of 50 μL, is as follows:

[0028] Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–25 μL nucleic acid sample, 10–30 U reverse transcriptase, 2–7 U Taq enzyme, 10–30 U RNase inhibitor; and use ddH2O to bring the reaction volume to 50 μL.

[0029] The second round of PCR amplification system, with a reaction volume of 50 μL, is as follows:

[0030] Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–15 μL of nucleic acid products from the first round of PCR amplification, 2–7 U Taq enzyme, and ddH2O to bring the reaction volume to 50 μL.

[0031] Furthermore, the PCR amplification procedure includes a first-round PCR amplification procedure and a second-round PCR amplification procedure; wherein,

[0032] The procedure for the first round of PCR amplification includes:

[0033] Reverse transcription for 40–50 min; pre-denaturation at 93–96℃ for 2–4 min; denaturation at 93–96℃ for 20–35 s, annealing at 53–60℃ for 40–50 s, extension at 70–75℃ for 1–2 min, for a total of 30–40 cycles; extension at 70–75℃ for 5–10 min.

[0034] The procedure for the second round of PCR amplification includes:

[0035] Pre-denaturation at 93–96℃ for 3–6 min; denaturation at 93–96℃ for 20–35 s, annealing at 53–60℃ for 40–50 s, and extension at 70–75℃ for 1–3 min, for a total of 3–4 cycles; denaturation at 93–96℃ for 20–35 s, annealing at 53–60℃ for 40–50 s, and extension at 70–75℃ for 1–3 min, for a total of 27–40 cycles; and further extension at 70–75℃ for 5–10 min.

[0036] Furthermore, during the sequencing of the nested PCR amplification products, the primers used are the sequencing primer set described in the second aspect.

[0037] Furthermore, the steps for analyzing the sequencing results include:

[0038] Open Sequencher 5.4.6 sequence assembly software, import the original sequence file; trim the original sequence; obtain the sequence to be analyzed;

[0039] Import the reference sequence template and set the reference sequence;

[0040] Select the sequence to be analyzed and the reference sequence, and perform comparative analysis;

[0041] Display peak diagrams and secondary peak diagrams, compare and analyze them to determine degenerate bases;

[0042] Drug resistance site alignment analysis was performed using Sequencher 5.4.6 sequence assembly software to obtain assembled sequences, which were assembled based on sequencing results and reflected the original base sequence; the assembled sequences were exported and saved.

[0043] Open the HIV DRUG RESISTANCE DATABASE website (https: / / hivdb.stanford.edu / hivdb / by-patterns / ), import the assembled sequences, and perform drug resistance analysis.

[0044] Fifthly, this application provides the application of the primer set described in the first aspect, the sequencing primer set described in the second aspect, the kit described in the third aspect, and / or the detection method described in the fourth aspect in detecting drug resistance mutations in the HIV-1 RNA integrase region.

[0045] Compared with the prior art, the present application adopts the above technical solution and has at least the following technical effects:

[0046] 1. In the prior art, when VL ≥ 1000 copies / mL, the success rate of drug resistance detection in the HIV-1 RNA integrase region is relatively high; while when VL is lower than 1000 copies / mL, the amplification failure rate of the HIV-1 viral gene is relatively high, which leads to the inability to effectively suppress and treat the condition of such patients in a timely manner. The technical solution provided by the present application can effectively solve the detection of drug resistance mutations in the HIV-1 RNA integrase region when VL < 1000 copies / mL.

[0047] 2. For the technical solution provided by the present application, by optimizing the drug resistance system, amplification procedure, primer sequences and the like, the drug resistance detection of the HIV-1 RNA integrase region is effectively improved, which not only enables the drug resistance detection of the HIV-1 RNA integrase region with VL ≥ 500 copies / mL to reach a 100% stable detection state, but also makes the success rate of the drug resistance detection of the HIV-1 RNA integrase increase with the rise of viral load when 0 copies / mL < VL < 500 copies / mL. Brief Description of the Drawings

[0048] Figure 1 shows the agarose gel electrophoresis identification result of successful drug resistance detection in the HIV-1 integrase region provided by the embodiment of the present application; wherein A is the analysis result of the PCR amplification product by agarose gel electrophoresis when the nucleic acid to be tested is DNA; B is the analysis result of the PCR amplification product by agarose gel electrophoresis when the nucleic acid to be tested is RNA.

[0049] Figure 2 shows the sequencing result analysis process diagram and the display diagram of bases determined as degenerate bases provided by the embodiment of the present application, wherein A is the degenerate base display diagram in the HIV-1 RNA sequencing result analysis, and it can be seen from the diagram that the degenerate base is Y(C / T); B is the degenerate base display diagram in the HIV-1 DNA sequencing result analysis, and it can be seen from the diagram that the degenerate base is R(A / G).

[0050] Figure 3The diagram shows the information on drug resistance sites and the results of drug resistance detection provided in the embodiments of this application. A is a schematic diagram of the region where the drug resistance site mutation site and the amplification product are located. As shown in the diagram, the amplification product obtained by this method contains the entire integrase region. B shows the mutation situation of the sequence of the amplified product in the integrase region. As shown in the diagram, the sequence mutates at amino acid 232 of the minor mutation site in the integrase region, and the amino acid changes from D to N. C is the contribution value of the minor mutation site in the integrase region shown in B to the drug resistance of integrase drugs. As shown in the diagram, D232N contributes 10 points to both EVG and RAL drugs, where 10 points represent the potential drug resistance of this sequence to both EVG and RAL drugs. Detailed Implementation

[0051] Detailed Description of Embodiments: The present application is further illustrated below with reference to specific embodiments. The described embodiments are only a portion of, and not all, of the embodiments of the present application. It should be understood that the following embodiments are provided to offer a complete disclosure and illustration to those skilled in the art on how to utilize the methods and compositions of the present application, and are not intended to limit the scope of the present application. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without inventive effort are within the scope of protection of the present application.

[0052] In one aspect, this application provides a primer set for amplifying the HIV-1 integrase region.

[0053] In this embodiment, the primer set includes: a first primer set, comprising a first set of upstream primers and a first set of downstream primers, wherein the first set of upstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 1-26; and the first set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 27-55; and a second primer set, comprising a second set of upstream primers and a second set of downstream primers, wherein the second set of upstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 56-82; and the second set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 83-112. In this embodiment, selecting at least one of the first set of upstream primers, the first set of downstream primers, the second set of upstream primers, and the second set of downstream primers is sufficient to amplify the HIV-1 integrase region, further for the purpose of detecting HIV-1 integrase region drug resistance.

[0054] In the embodiments of this application, in the primer sequence of the primer set, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters R / D / V / Y / N / B represent mixed bases; "mixed bases" means that two or three bases are present at the position, which can capture and amplify sequences that have mutated at a specified position; wherein, R represents A+G; D represents A+G+T; V represents A+G+C; Y represents C+T; N represents A+G+C+T; and B represents C+G+T.

[0055] In the embodiments of this application, some primers in the primer set contain at least one of the above-mentioned mixed bases. The first set of upstream primers includes four mixed bases: R, D, V, and Y, such as SEQ ID NO: 1 containing R and D, SEQ ID NO: 2 containing V and Y, and SEQ ID NO: 3 containing R and Y. The first set of downstream primers includes one mixed base: N, such as SEQ ID NO: 44 containing N. The second set of upstream primers includes three mixed bases: R, V, and B, such as SEQ ID NO: 61 containing R, SEQ ID NO: 70 containing B, and SEQ ID NO: 77 containing V. The second set of downstream primers includes two mixed bases: Y and R, such as SEQ ID NO: 85 containing Y and SEQ ID NO: 109 containing R.

[0056] In some embodiments, the first set of upstream primers in the primer set for amplifying the HIV-1 integrase region includes at least N primers with nucleotide sequences of SEQ ID NO: 1 to 26, wherein N is any natural number from 2 to 26; the first set of downstream primers includes at least W primers with nucleotide sequences of SEQ ID NO: 26 to 55, wherein W is any natural number from 2 to 30; the second set of upstream primers includes at least T primers with nucleotide sequences of SEQ ID NO: 56 to 82, wherein T is any natural number from 2 to 27; and the second set of downstream primers includes at least Q primers with nucleotide sequences of SEQ ID NO: 83 to 112, wherein Q is any natural number from 2 to 30.

[0057] In a preferred embodiment, the first set of upstream primers in the primer set for amplifying the HIV-1 integrase region includes all primers with nucleotide sequences SEQ ID NO: 1–26; the first set of downstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 27–55; the second set of upstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 56–82; and the second set of downstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 83–112.

[0058] Secondly, this application provides a sequencing primer set.

[0059] In this embodiment of the application, the sequencing primer set is used to sequence the amplified product of the HIV-1 integrase region gene, and the amplified product is obtained by PCR amplification using the aforementioned primer set; the sequencing primer set includes at least four primers with nucleotide sequences of SEQ ID NO: 113-138, and the sequencing primer set includes forward sequencing primers and reverse sequencing primers.

[0060] In the embodiments of this application, in the sequencing primer sequences, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters R / D represent mixed bases; wherein, R represents A+G; and D represents A+G+T.

[0061] In the embodiments of this application, some sequences of the sequencing primers contain R, such as SEQ ID NO: 116 which contains R; and some sequences contain D, such as SEQ ID NO: 129 and SEQ ID NO: 138 which both contain D.

[0062] In some embodiments, the sequencing primer set includes at least S types of primers with nucleotide sequences of SEQ ID NO: 113-138, where S is any natural number from 5 to 26.

[0063] In a preferred embodiment, the sequencing primer set includes all 26 primers with nucleotide sequences SEQ ID NO: 113-138.

[0064] Thirdly, this application provides a kit for detecting drug resistance mutations in the integrase region of HIV-1 RNA, the kit comprising RNA extraction reagent, RNA reverse transcription reagent, the primer set described in the first aspect and / or the sequencing primer set described in the second aspect.

[0065] Furthermore, the kit is used to detect drug resistance mutations in the integrase region of HIV-1 RNA, and the nucleic acid sample is RNA.

[0066] Fourthly, this application provides a method for detecting drug resistance mutations in the HIV-1 RNA integrase region.

[0067] In this embodiment of the application, the detection method includes: extracting nucleic acid from the sample to be tested; performing nested PCR amplification on the previously obtained nucleic acid using the aforementioned primer set; performing agarose gel electrophoresis analysis on the nested PCR amplification product; sequencing the nested PCR amplification product; and analyzing the sequencing results to obtain the drug resistance mutation result of the HIV-1 RNA integrase region.

[0068] In this embodiment, if the nucleic acid of the sample to be tested is DNA, then the extracted DNA is used directly as a template for PCR amplification; if the nucleic acid of the sample to be tested is RNA, then the aforementioned method further includes a step of reverse transcribing RNA into cDNA and using cDNA as a template for amplification.

[0069] In this embodiment of the application, the nested PCR amplification includes: a first round of PCR amplification, wherein the primers used for the first round of PCR amplification are the aforementioned first primer set; and a second round of PCR amplification, wherein the primers used for the second round of PCR amplification are the aforementioned second primer set.

[0070] In this embodiment, the PCR amplification system includes: a first-round PCR amplification system, with a reaction volume of 50 μL, specifically: Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–25 μL nucleic acid sample, 10–30 U reverse transcriptase, 2–7 U Taq enzyme, 10–30 U RNase inhibitor, and ddH2O to bring the reaction volume to 50 μL; and the second round PCR amplification system, with a reaction volume of 50 μL, specifically: Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–15 μL of nucleic acid products from the first round of PCR amplification, 2–7 U Taq enzyme, and ddH2O to bring the reaction volume to 50 μL.

[0071] In this embodiment, the PCR amplification program includes a first-round PCR amplification program and a second-round PCR amplification program; wherein, the first-round PCR amplification program includes: an RNA amplification program, specifically: reverse transcription for 40-50 min; pre-denaturation at 93-96℃ for 2-4 min; denaturation at 93-96℃ for 20-35 s, annealing at 53-60℃ for 40-50 s, extension at 70-75℃ for 1-2 min, for a total of 30-40 cycles; and a further extension at 70-75℃ for 5-10 min; and / or a DNA amplification program, pre-denaturation at 93-96℃ for 2-4 min; denaturation at 93-96℃ for 20-35 s. The first round of PCR amplification program includes: annealing at 53-60℃ for 40-50s, extension at 70-75℃ for 1-2min, for a total of 37-40 cycles; and a further extension at 70-75℃ for 5-10min. The second round of PCR amplification program includes: pre-denaturation at 93-96℃ for 3-6min; denaturation at 93-96℃ for 20-35s, annealing at 53-60℃ for 40-50s, extension at 70-75℃ for 1-3min, for a total of 3-4 cycles; denaturation at 93-96℃ for 20-35s, annealing at 53-60℃ for 40-50s, extension at 70-75℃ for 1-3min, for a total of 27-40 cycles; and a further extension at 70-75℃ for 5-10min.

[0072] In this embodiment of the application, the aforementioned sequencing primer set is used during the sequencing of nested PCR amplification products.

[0073] In some preferred embodiments, the first set of upstream primers in the primer set used for amplifying the HIV-1 integrase region includes all primers with nucleotide sequences SEQ ID NO: 1–26; the first set of downstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 27–55; the second set of upstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 56–82; the second set of downstream primers in the primer set includes all primers with nucleotide sequences SEQ ID NO: 83–112; the selected sequencing primer set includes all 26 primers with nucleotide sequences SEQ ID NO: 113–138; the specific process of applying the selected detection method to the detection of drug resistance mutations in the HIV-1 RNA integrase region is as follows:

[0074] 1. Nucleic acid extraction:

[0075] This method requires the extraction of two different nucleic acids, DNA and RNA, from anticoagulated whole blood, PBMC cells, dried blood spots, and serum / plasma, and the detection of nucleic acid concentrations using two different methods.

[0076] (1) DNA nucleic acid extraction (80 samples)

[0077] Take 200 μL of sample, add 200 μL of lysis buffer and 30 μL of enzyme K solution, vortex to mix for 15 s, incubate at 56 °C for 10 min, then briefly centrifuge; add 200 μL of anhydrous ethanol, vortex to mix for 15 s; briefly centrifuge, transfer all to adsorption column A, centrifuge at 8000 rpm for 1 min, discard the 2 mL collection tube, and transfer adsorption column A to a new 2 mL collection tube A; add 500 μL of wash buffer 1, centrifuge at 8000 rpm for 1 min, discard the 2 mL collection tube A, and transfer adsorption column A to a new 2 mL collection tube A; add 500 μL of wash buffer 2, centrifuge at 13000 rpm for 3 min, discard the 2 mL collection tube A, and transfer adsorption column A to a new 2 mL collection tube A; centrifuge at 13000 rpm for 1 min, discard the 2 mL collection tube A, and transfer adsorption column A to a new 1.5 mL collection tube A. Add 60 μL of sulfacetamide (SA) solution to an EP tube, let stand for 1 min, centrifuge at 8000 rpm for 1 min, and collect 1.5 mL of the EP tube as the extracted nucleic acid product. For short-term storage, store at -20℃; for long-term storage, store below -70℃.

[0078] DNA nucleic acid extraction results: The concentration of the extracted nucleic acid was determined using nanodrop, and the concentration values ​​are shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] The concentration of HIV-1 DNA was quantitatively detected using real-time quantitative PCR, and the results are shown in Table 2.

[0083] Table 2

[0084]

[0085]

[0086] As can be seen from Table 2, the selected samples included both those with high HIV-1 DNA levels and those with levels below 500 copies / mL.

[0087] (2) RNA nucleic acid extraction (80 samples)

[0088] Add 800 μL of lysis buffer and 5.6 μL of carrier RNA to 300 μL of serum / plasma sample, vortex to mix for 30 s, and incubate at room temperature for 10 min. Add 300 μL of isopropanol, mix by inverting for 30 seconds, and incubate at room temperature for 2 minutes. Centrifuge briefly, then transfer 710 μL to a centrifugal adsorption column and centrifuge at 10,000 rpm for 1 minute. Discard the 2 mL collection tube and transfer the centrifugal adsorption column to a new 2 mL collection tube. Repeat twice until all samples are transferred to the centrifugal adsorption column. Discard the 2 mL collection tube and transfer the centrifugal adsorption column to a new 2 mL collection tube. Add 500 μL of wash buffer 1 and centrifuge at 10,000 rpm for 1 minute. Discard the 2 mL collection tube and transfer the centrifugal adsorption column to a new 2 mL collection tube. Add 500 μL of wash buffer 2 and centrifuge at 14,000 rpm for 3 minutes. Discard the 2 mL collection tube and transfer the centrifugal adsorption column to a new 2 mL collection tube and centrifuge at 14,000 rpm for 1 minute. Discard the 2 mL collection tube and transfer the centrifugal adsorption column to a new 1.5 mL collection tube. Open the EP tube, let it stand for 2 minutes, add 60 μL of elution buffer (TE), let it stand for 2 minutes, centrifuge at 10000 rpm for 1 minute, and collect 1.5 mL of EP tube as the extracted nucleic acid sample. Store the extracted nucleic acid sample at -80℃ and avoid repeated freezing.

[0089] RNA nucleic acid extraction results: The concentration of the extracted nucleic acid was determined using nanodrop, and the concentration values ​​are shown in Table 3.

[0090] Table 3

[0091]

[0092]

[0093] The concentration of HIV-1 RNA was quantitatively detected using real-time quantitative PCR, and the results are shown in Table 4.

[0094] Table 4

[0095]

[0096] As can be seen from the table, the selected samples included those with high HIV-1 RNA levels as well as those with levels below 1000 copies / mL. Existing national guidelines indicate that the drug resistance success rate for samples with levels >1000 copies / mL is around 85%.

[0097] 2. First round of nucleic acid PCR amplification:

[0098] (1) Preparation of reaction system: Mg 2+ 6 μM PCR buffer, 1 μM primers (there are 55 primers, the nucleotide sequences of which are shown in SEQ ID NO: 1-55), 0.75 μM dNTPs, 25 U reverse transcriptase, 3 U Taq enzyme, 25 U RNase inhibitor. The volume of the reaction system is made up to 30 μL using ddH2O (if the nucleic acid type of the sample is DNA, there is no need to add RNase inhibitor and reverse transcriptase, and the volume is made up with ddH2O).

[0099] (2) Calculate the required amount of the reaction solution for this experiment based on the single-person reaction solution system (the loss value should be included), and dispense 30 μL into each PCR eight-pack.

[0100] (3) Add 20 μL of the extracted and purified nucleic acid sample, label it, vortex for 30 seconds, and then centrifuge quickly.

[0101] (4) PCR amplification: After inserting the eight-piece array into the instrument, select the following PCR amplification program:

[0102] A. When the nucleic acid type of the sample is RNA: reverse transcription at 45℃ for 40 min; pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 25 s, annealing at 57℃ for 40 s, extension at 72℃ for 1 min, for a total of 37 cycles; extension at 72℃ for 7 min.

[0103] B. When the nucleic acid type of the sample is DNA: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 25 s, 57℃ annealing for 40 s, 72℃ extension for 1 min, for a total of 37 cycles; 72℃ extension for 7 min;

[0104] The experiment lasted approximately 3 hours.

[0105] 3. Second round of nucleic acid PCR amplification:

[0106] (1) Preparation of reaction system: Mg 2+ 6 μM PCR buffer, 1 μM primers (there are 57 primers, and their nucleotide sequences are shown in SEQ ID NO: 56-112), 0.75 μM dNTPs, 3 U Taq enzyme, and ddH2O to bring the volume of the reaction system to 40 μL.

[0107] (2) Calculate the required amount of the reaction solution for this experiment based on the single-person reaction solution system (the loss value should be included), and dispense 40 μL into each PCR eight-pack.

[0108] (3) Add 10 μL of the extracted and purified nucleic acid sample, label it, vortex for 30 seconds, and then centrifuge quickly.

[0109] (4) PCR amplification: After inserting the eight-piece array into the instrument, select the following PCR amplification program:

[0110] A. When the nucleic acid type of the sample is RNA: reverse transcription at 45℃ for 40 min; pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 25 s, annealing at 57℃ for 40 s, extension at 72℃ for 1 min, for a total of 37 cycles; extension at 72℃ for 7 min.

[0111] B. When the nucleic acid type of the sample is DNA: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 25 s, 57℃ annealing for 40 s, 72℃ extension for 1 min, for a total of 37 cycles; 72℃ extension for 7 min;

[0112] The experiment lasted approximately 3 hours.

[0113] 4. Agarose gel electrophoresis was used to analyze the PCR amplification products of RNA and DNA samples, respectively:

[0114] TAE dilution: 10 mL (50x TAE) + 490 mL pure water to obtain 1×TAE; prepare 2% agarose, the specific amount depends on the size of the prepared gel; dissolve in a microwave oven; cool the agarose solution to about 65°C at room temperature, add nucleic acid dye, shake well and pour into an electrophoresis tank to cool into a gel; after the gel solidifies; transfer the gel to the electrophoresis tank for sample loading: the sample volume is 10 μL, with a 2 kb marker added in the middle; perform agarose gel electrophoresis, and take pictures and save them after electrophoresis; mark the samples with the target band on the table.

[0115] Agarose gel electrophoresis analysis results: such as Figure 1As shown in Table 5, A represents the analysis results of PCR amplification products by agarose gel electrophoresis when the target nucleic acid is RNA; B represents the analysis results of PCR amplification products by agarose gel electrophoresis when the target nucleic acid is DNA. The target band is between 1000 and 2000 bp; the presence of a band indicates successful amplification, allowing for subsequent drug resistance testing. The detection results are shown in Table 5. The detection method provided in this application achieves a 100% detection rate for HIV-1 DNA samples with a concentration greater than 200 copies / mL and HIV-1 RNA samples with a concentration greater than 500 copies / mL, and its success rate across all concentration ranges is significantly higher than existing technologies.

[0116] Table 5

[0117]

[0118]

[0119] 5. Sequencing and analysis of PCR amplification products were performed, and the amplification products of RNA and DNA samples were analyzed separately:

[0120] (1) Sequencing of PCR amplification products: The target band of the above PCR amplification was cut out and purified;

[0121] The concentration of the recovered product was determined using an ND8000 measuring instrument, and the amount of sample loaded for subsequent sequencing reactions was determined by conversion. After calculating the volume of water, betaine, primers, template, and BDT added based on the concentration, the mixture was centrifuged at 1200 rpm for 30 seconds to carry out the BDT reaction.

[0122] After the BDT reaction experiment, the sample needs to be adsorbed with magnetic beads, and the remaining unreacted components are washed away with 85% alcohol. Finally, sterile water is added, and the sample is placed on a sequencer for sequencing. The nucleotide sequences of the sequencing primers are shown in SEQ ID NO: 113-138.

[0123] (2) Post-sequencing data analysis:

[0124] Open Sequencher 5.4.6 sequence assembly software, click File→Import→Folder of sequences, select all the raw files generated by sequencing to import, and perform sequence alignment; display the peak diagram, and align according to drug resistance sites to see if gene mutations have occurred, such as... Figure 2 As shown; after completion, save the project and export the assembled sequence to the specified folder; open the HIV DRUG RESISTANCE DATABASE drug resistance analysis website, analyze the mutation status of the sequence, and calculate the information on drug resistance sites and the degree of drug resistance, such as... Figure 3 As shown.

[0125] This method differs from existing technologies in its post-sequencing data processing. First, it removes poor-quality sequence heads and tails, such as those with excessive sequencing background peaks or unclear base identification. Second, it defines a degenerate base at a site as one where the height of the secondary peak is more than 30% of the height of the main peak.

[0126] Subtype analysis:

[0127] The concatenated sequence is used to determine the subtype in a phylogenetic tree manner. This method can currently identify subtypes B, C, D, CRF01_AE, A1, G, CPZ, CRF02_AG, CRF37_CPX, CRF03_AB, CRF36_06A1, CRF07_BC, CRF08_BC, CRF71_BF1, CRF12_BF, F1, CRF21_A2D, CRF50_A1D, CRF55_01B, CRF05_DF, CRF44_BF, and CRF49_C. PX, CRF78_CPX, CRF96_CPX, CRF06_CPX, CRF09_CPX, CRF11_CPX, CRF13_CPX, CRF14_BG, CRF15_01B, CRF16_A2D, C RF18_CPX, CRF19_CPX, CRF20_BG, CRF04_CPX, CRF22_01A1, CRF23_BG, CRF24_BG, CRF25_CPX, CRF26_A5U, CRF27_C PX, CRF29_BF1, CRF31_BC, CRF33_01B, CRF34_01B, CRF35_A1D, CRF36_CPX, CRF37_CPX, CRF38_BF1, CRF39_BF1, C RF40_BF1, CRF43_02G, CRF47_BF1, CRF48_01B, CRF51_01B, CRF53_01B, CRF54_01B, CRF56_CPX, CRF57_BC, CRF58_ Drug resistance testing for multiple HIV-1 subtypes, including CRF59_01B, CRF61_BC, CRF62_BC, CRF64_BC, CRF65_CPX, CRF69_01B, CRF70_BF1, CRF72_BF1, CRF73_BG, CRF74_01B, CRF77_CPX, CRF79_0107, CRF82_CPX, CRF83_CPX, CRF85_BC, CRF86_BC, CRF90_BF1, and subtype O.

[0128] 6. Test Results and Analysis

[0129] Figure 2 This is a diagram illustrating the degenerate bases, and also shows their sequencing peak results. Figure 2 In A, we can see that position 537 is mutated from T to the degenerate base Y (C / T); from Figure 2 In B, we can see that at position 531, A has mutated into the degenerate base R (A / G); and where such positions are present, a bimodal pattern is observed, with the secondary peak being more than 30% of the height of the primary peak.

[0130] Figure 3 This image shows information on drug resistance sites and the results of drug resistance testing. Figure 3 A shows the mutation map and location of drug resistance sites; Figure 3 B represents the minor mutation site in the integrase region at position 232, where a D mutation was changed to a N mutation. Figure 3 C refers to the contribution of EVG and RAL to the resistance site at the D232N site, where each drug contributes 10%. The degree of viral resistance to each drug can be determined based on its score.

[0131] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A primer set for amplifying the HIV-1 RNA integrase region, characterized in that, The primer set includes: The first primer set comprises a first set of upstream primers and a first set of downstream primers. The first set of upstream primers comprises all primers with nucleotide sequences of SEQ ID NO: 1–26; the first set of downstream primers comprises all primers with nucleotide sequences of SEQ ID NO: 27–55; and The second primer set includes a second set of upstream primers and a second set of downstream primers. The second set of upstream primers includes all primers with nucleotide sequences of SEQ ID NO: 56-82; the second set of downstream primers includes all primers with nucleotide sequences of SEQ ID NO: 83-112.

2. A kit for detecting drug resistance mutations in the integrase region of HIV-1 RNA, characterized in that, The kit includes RNA extraction reagent, RNA reverse transcription reagent, the primer set as described in claim 1, and sequencing primer set; the sequencing primer set includes primers with nucleotide sequences of SEQ ID NO: 113-138.

3. A method for detecting drug resistance mutations in the HIV-1 RNA integrase region, which is not a disease diagnosis or treatment method, characterized in that... The detection method includes: Total RNA was extracted from the sample to be tested, and the RNA was reverse transcribed into cDNA. Nested PCR amplification was performed using cDNA as a template and the primer set described in claim 1 to obtain nested PCR amplification products. Electrophoretic analysis of the nested PCR amplification products was performed to confirm the target band; The nested PCR amplification products were purified and then sequenced using the sequencing primers described in claim 2; and The sequencing results were analyzed to obtain the drug resistance mutation results of the HIV-1 RNA integrase region.

4. The detection method according to claim 3, characterized in that, The nested PCR amplification includes: The first round of PCR amplification uses the first primer set from the primer set described in claim 1. The second round of PCR amplification uses the second primer set from the primer set described in claim 1.

5. The detection method according to claim 4, characterized in that, The reaction volume for the first round of PCR amplification is calculated to be 50 μL, specifically as follows: Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–25 μL nucleic acid sample, 10–30 U reverse transcriptase, 2–7 U Taq enzyme, 10–30 U RNase inhibitor, and ddH2O to bring the reaction volume to 50 μL. The reaction volume for the second round of PCR amplification is calculated to be 50 μL, specifically as follows: Mg 2+ 4–7 μM PCR buffer, 0.5–1 μM primers each, 0.5–1 μM dNTPs, 5–15 μL of nucleic acid products from the first round of PCR amplification, 2–7 U Taq enzyme, and ddH2O to bring the reaction volume to 50 μL.

6. The detection method according to claim 5, characterized in that, The procedure for the first round of PCR amplification includes: Reverse transcription for 40–50 min; pre-denaturation at 93–96℃ for 2–4 min; denaturation at 93–96℃ for 20–35 s, annealing at 53–60℃ for 40–50 s, extension at 70–75℃ for 1–2 min, for a total of 20–40 cycles; extension at 70–75℃ for 5–10 min. The procedure for the second round of PCR amplification includes: Pre-denaturation at 93-96℃ for 3-6 min; denaturation at 93-96℃ for 20-35 s, annealing at 53-60℃ for 40-50 s, and extension at 70-73℃ for 1-3 min, for a total of 3-4 cycles; denaturation at 93-96℃ for 20-35 s, annealing at 53-60℃ for 40-50 s, and extension at 70-73℃ for 1-3 min, for a total of 27-40 cycles; and further extension at 70-75℃ for 5-10 min.

7. The use of the primer set of claim 1 and the kit of claim 2 in the preparation of products for detecting drug resistance mutations in the integrase region of HIV-1 RNA.

8. The application according to claim 7, characterized in that, The sample types selected for detecting drug resistance mutations in the HIV-1 RNA integrase region are any one of serum, plasma, semen, urine, and cerebrospinal fluid, and the extracted nucleic acid is RNA.

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