Primer set and method for detecting drug resistance mutation in HIV-1 RNA protease and reverse transcriptase region

Abstract

The application discloses a primer group and a method for detecting HIV-1 RNA protease and reverse transcriptase region drug resistance site mutation. The detection method can be applied to protease and reverse transcriptase drug resistance detection of HIV-1 RNA including low-concentration HIV-1 RNA by optimizing a drug resistance reaction system, an amplification program and primer sequences. The detection method can use serum, plasma, semen, urine, cerebrospinal fluid and other human body fluids as samples, adopts a specific primer group, amplifies HIV-1 protease and reverse transcriptase region sequences through a nested PCR method, and determines the amplified sequences through a Sanger sequencing method to analyze drug resistance related gene mutations, so that the HIV-1 RNA protease and reverse transcriptase region drug resistance detection efficiency is greatly improved. Whether the patient is resistant to a specific drug and the degree of drug resistance are judged by using a genotype drug resistance interpretation system, so as to provide a reference basis for the patient to formulate 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 detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions, a method for detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions using the primer set, and a detection kit for HIV-1 RNA protease and reverse transcriptase region drug resistance site mutations containing the primer set. Background Technology

[0002] Human immunodeficiency virus (HIV) infection is the primary cause of AIDS (acquired immune deficiency syndrome). It is estimated that in 2020, approximately 37.7 million people worldwide were infected with HIV, with 680,000 deaths, and about 1.5 million new infections each year. Due to its rapid transmission, the lack of a cure, and its severe consequences, it has received widespread global attention.

[0003] Highly Active Antiretroviral Therapy (HAART), also known as combination antiretroviral therapy (cART), is currently the only antiretroviral therapy approved by the FDA. It is a method that combines three or more antiretroviral drugs to treat acquired immunodeficiency syndrome. This method can effectively inhibit HIV replication, reduce viral load, and restore or partially restore the damaged immune function of the body. In China, highly effective antiretroviral therapy for HIV infection mainly involves the combined use of three classes of drugs: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). NRTIs, after phosphorylation within cells, generate active metabolites that competitively inhibit the binding of natural nucleosides to HIV reverse transcriptase, thus hindering HIV cDNA production and inhibiting HIV replication. NNRTIs, through non-competitive binding to HIV reverse transcriptase, inhibit viral replication, thereby producing an antiviral effect. PIs competitively block the binding of HIV proteases to their natural substrates, inhibiting late-stage HIV replication.

[0004] 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. Routine drug resistance testing has a success rate of approximately 90% when the viral load is >1000 copies / mL. Therefore, clinicians generally believe that HIV-1 RNA resistance testing should only be performed when the viral load exceeds 1000 copies / mL. However, due to the use of highly effective antiretroviral drugs, this group represents a relatively small percentage of patients clinically. Patients with low viral loads of 50-1000 copies / mL account for approximately 30% of clinical cases, and studies have shown that LLV is associated with HIV-1 drug resistance.

[0005] Therefore, in order to meet market demand, there is an urgent need to develop a method for detecting drug resistance mutations in low-concentration protease and reverse transcriptase regions of RNA. Summary of the Invention

[0006] To address the problems of existing technologies, this application provides a method for detecting drug resistance in HIV-1 RNA protease and reverse transcriptase regions. This method can be applied to the detection of drug resistance in all concentrations of HIV-1 RNA, including low concentrations of protease and reverse transcriptase. The method uses human body fluids such as serum, plasma, semen, urine, and cerebrospinal fluid as samples. PCR and Sanger sequencing are used to determine the gene fragment sequences of the HIV-1 RNA protease and reverse transcriptase regions. The obtained gene fragment sequences are compared with wild-type gene sequences and the sequence information of 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 can provide a reference for patients to develop first-line antiviral treatment, second-line treatment plans, pre- and post-exposure prophylaxis regimens, and measures to reduce the occurrence of drug resistance.

[0007] In a first aspect, this application provides a primer set for amplifying the HIV-1 RNA protease and reverse transcriptase regions, the primer set comprising:

[0008] 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 27; and the first set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 28 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 80; and the second set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 81 to 110.

[0009] Furthermore, in the primer sequence of the primer set, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters H / R / Y represent degenerate (mixed) bases; wherein, H represents A+T+C, R represents A+G, and Y represents C+T.

[0010] Furthermore, the first set of upstream primers includes two degenerate bases, H and R; the first set of downstream primers includes two degenerate bases, R and Y; the second set of upstream primers includes two degenerate bases, R and Y; and the second set of downstream primers includes two degenerate bases, Y and R.

[0011] Secondly, this application provides a sequencing primer set for sequencing the amplified products of HIV-1 protease and reverse transcriptase regions, wherein the amplified products are 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: 111-134, and the sequencing primer set includes forward sequencing primers and reverse sequencing primers.

[0012] Furthermore, in the sequencing primer sequence, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; the letters R / Y represent degenerate bases; where R represents A+G and Y represents C+T.

[0013] Thirdly, this application provides a kit for detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions, 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.

[0014] Fourthly, this application provides a method for detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions, the detection method comprising:

[0015] Total RNA was extracted from the sample to be tested, and the RNA was reverse transcribed into cDNA.

[0016] 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;

[0017] The nested PCR amplification products were analyzed by electrophoresis (e.g., agarose gel electrophoresis).

[0018] The nested PCR amplification products were purified and then sequenced; and

[0019] The sequencing results were analyzed to obtain the drug resistance mutation results in the HIV-1 RNA protease and reverse transcriptase regions.

[0020] Furthermore, the nested PCR amplification includes:

[0021] The first round of PCR amplification uses the aforementioned first primer set as the primers.

[0022] The second round of PCR amplification used the same primers as the aforementioned second primer set.

[0023] Furthermore, the primers used for sequencing the amplified products are the sequencing primer set described in the second aspect.

[0024] Furthermore, the PCR amplification system includes:

[0025] The first round of PCR amplification system, with a reaction volume of 50 μL, is as follows:

[0026] Mg 2+ 5–15 μL (stock solution concentration: 25 mM), 5× buffer 5–15 μL, forward and reverse primers 0.5–1.5 μL each (stock solution concentration: 20 μM), dNTP 0.5–1 μL (stock solution concentration: 100 mM), RNA nucleic acid sample 5–25 μL, reverse transcriptase 0.1–0.3 μL (stock solution concentration: 160 U / uL), Taq enzyme 0.5–1 μL (stock solution concentration: 5 U / uL), RNase inhibitor 0.3–0.7 μL (stock solution concentration: 40 U / uL), and ddH2O to bring the reaction volume to 50 μL.

[0027] The reaction volume for the second round of PCR amplification is calculated to be 50 μL, specifically as follows:

[0028] Mg 2+5–15 μL (stock solution concentration: 25 mM), 5–15 μL 5× buffer, 0.5–1.5 μL each of forward and reverse primers (stock solution concentration: 20 μM), 0.5–1 μL dNTP (stock solution concentration: 100 mM), 5–15 μL of nucleic acid product from the first round of PCR amplification, 0.5–1 μL Taq enzyme (stock solution concentration: 5 U / μL), and use ddH2O to bring the volume of the reaction system to 50 μL.

[0029] Furthermore, the PCR amplification procedure includes a first-round PCR amplification procedure and a second-round PCR amplification procedure; wherein,

[0030] The procedure for the first round of PCR amplification includes:

[0031] Reverse transcription at 50℃ for 40–50 min; pre-denaturation at 93–96℃ for 1–3 min; denaturation at 93–96℃ for 20–35 s, annealing at 45–55℃ for 25–35 s, extension at 70–75℃ for 1–3 min, for a total of 25–40 cycles; further extension at 70–75℃ for 5–10 min, and storage at 4℃.

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

[0033] Pre-denaturation at 93-96℃ for 3-5 min; denaturation at 93-96℃ for 10-20 s, annealing at 50-60℃ for 15-30 s, extension at 70-75℃ for 1-3 min, for a total of 30-45 cycles; further extension at 70-75℃ for 5-10 min, and storage at 4℃.

[0034] Furthermore, during the sequencing of the nested PCR amplification products, the primers used are the sequencing primer set described in the second aspect.

[0035] Furthermore, the steps for analyzing the sequencing results include:

[0036] Open Sequencher 5.4.6 sequence assembly software, import the original sequence file; trim the original sequence; obtain the sequence to be analyzed;

[0037] Import the reference sequence template and set the reference sequence;

[0038] Select the sequence to be analyzed and the reference sequence, and perform comparative analysis;

[0039] Display peak diagrams and secondary peak diagrams, compare and analyze them to determine degenerate bases;

[0040] 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.

[0041] Open the HIV DRUG RESISTANCE DATABASE website (https: / / hivdb.stanford.edu / hivdb / by-patterns / ), import the assembled sequences, and perform drug resistance analysis.

[0042] 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 site mutations in the HIV-1 RNA protease and reverse transcriptase regions.

[0043] Compared with the prior art, the technical solution adopted in this application has at least the following technical effects:

[0044] 1. Currently, when VL ≥ 1000 copies / mL, the success rate of detecting HIV-1 RNA protease and reverse transcriptase resistance is relatively high; however, when VL is below 1000 copies / mL, the amplification failure rate of the HIV-1 viral gene is high. This results in some patients having undetectable or overlooked HIV resistance at excessively low concentrations, making it difficult for clinicians to effectively suppress the disease and adjust treatment plans in a timely manner. This is also a pressing issue that patients urgently want to understand.

[0045] 2. This application significantly improves the detection efficiency of HIV-1 RNA protease and reverse transcriptase regions by optimizing the drug resistance reaction system, amplification procedure, and primer sequences. It not only stabilizes the detection of HIV-1 RNA protease and reverse transcriptase region resistance in patients with a viral load ≥ 500 copies / mL, but also increases the success rate of detection for low viral load HIV-1 RNA protease and reverse transcriptase region resistance in patients with a viral load < 500 copies / mL, with the success rate increasing with increasing viral load. Attached Figure Description

[0046] Figure 1 The agarose gel electrophoresis results show the successful detection of HIV-1 protease and reverse transcriptase resistance in the embodiments of this application; the nucleic acid to be tested is RNA.

[0047] Figure 2 This is an information diagram of the drug resistance site provided in the embodiments of this application, namely, a schematic diagram of the drug resistance site mutation site of HIV-1 RNA and the region where the amplification product is located. The diagram shows the mutation status of the sequence in its respective enzyme region; for example, K20R represents a mutation at the 20th amino acid in the protease region, where the amino acid changes from K to R. Similarly, the mutation status of all bases in the region can be obtained from the diagram.

[0048] Figure 3The phylogenetic tree diagram provided in this application embodiment is shown in which the bolded part represents the standard sequence of different subtypes, the test samples are named with numbers, and the subtype analysis of unknown samples is performed by using the phylogenetic tree model. Detailed Implementation

[0049] Detailed Implementation: The present application is further illustrated below with reference to specific embodiments. The described embodiments are only some, not all, of the embodiments of the present application. It should be understood that the examples provided are intended to illustrate the application.

[0050] The following embodiments are provided to give a complete disclosure and illustration to those skilled in the art on how to utilize the methods and compositions of this application, and are not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0051] Firstly, this application provides a primer set for amplifying the HIV-1 protease and reverse transcriptase regions. In embodiments of this application, 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: 1–27; the first set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 28–55; 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: 56–80; and the second set of downstream primers comprises at least one primer with nucleotide sequences of SEQ ID NO: 81–110. In embodiments of this application, 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 protease and reverse transcriptase regions, and further for detecting drug resistance in the HIV-1 protease and reverse transcriptase regions.

[0052] 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 H / R / Y represent degenerate bases; "degenerate bases" means that there are two, three, or four bases mixed at this position, which can capture and amplify sequences that have mutated at a specified position; wherein, H represents A+C+T, R represents A+G, and Y represents C+T.

[0053] In the embodiments of this application, some primers in the primer set contain at least one of the aforementioned degenerate bases. Specifically, the first set of upstream primers includes both H and R degenerate bases, such as H in SEQ ID NO: 1, R in SEQ ID NO: 7, and R in SEQ ID NO: 12; the first set of downstream primers includes both R and Y degenerate bases, such as R in SEQ ID NO: 33 and Y in SEQ ID NO: 44; the second set of upstream primers includes both R and Y degenerate bases, such as R in SEQ ID NO: 56 and 75, and Y in SEQ ID NO: 65 and 69; the second set of downstream primers includes both Y and R degenerate bases, such as R in SEQ ID NO: 81, 82, and 102, and Y in SEQ ID NO: 84, 86, and 95.

[0054] In some embodiments, the first set of upstream primers in the primer set for amplifying the HIV-1 protease and reverse transcriptase regions comprises at least N primers with nucleotide sequences of SEQ ID NO: 1 to 27, wherein N is any natural number from 2 to 27; the first set of downstream primers comprises at least W primers with nucleotide sequences of SEQ ID NO: 27 to 55, wherein W is any natural number from 2 to 29; the second set of upstream primers comprises at least T primers with nucleotide sequences of SEQ ID NO: 56 to 80, wherein T is any natural number from 2 to 25; and the second set of downstream primers comprises at least Q primers with nucleotide sequences of SEQ ID NO: 81 to 110, wherein Q is any natural number from 2 to 30.

[0055] In a preferred embodiment, the first set of upstream primers in the primer set for amplifying the HIV-1 protease and reverse transcriptase regions includes all primers with nucleotide sequences of SEQ ID NO: 1–27; the first set of downstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 28–55; the second set of upstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 56–80; and the second set of downstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 81–110.

[0056] Secondly, this application provides a sequencing primer set.

[0057] In this embodiment of the application, the sequencing primer set is used to sequence the amplified products of HIV-1 protease and reverse transcriptase regions, and the amplified products are 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: 111-134, and the sequencing primer set includes forward sequencing primers and reverse sequencing primers.

[0058] 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 / Y represent degenerate bases; wherein, R represents A+G; and Y represents C+T.

[0059] In the embodiments of this application, some sequences of the sequencing primers contain R, such as R in SEQ ID NO: 112; some sequences contain Y, such as Y in SEQ ID NO: 120.

[0060] In some embodiments, the sequencing primer set includes at least S types of primers with nucleotide sequences of SEQ ID NO: 111-134, where S is any natural number from 5 to 24.

[0061] In a preferred embodiment, the sequencing primer set includes all 24 primers with nucleotide sequences SEQ ID NO: 111-134.

[0062] Thirdly, this application provides a kit for detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions, 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.

[0063] Furthermore, the kit is used to detect RNA nucleic acid samples for detecting drug resistance site mutations in the HIV-1 RNA protease and reverse transcriptase regions.

[0064] Fourthly, this application provides a method for detecting drug resistance mutations in the HIV-1 RNA protease and reverse transcriptase regions.

[0065] 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 drug resistance mutation results in the HIV-1 RNA protease and reverse transcriptase regions.

[0066] 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.

[0067] 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.

[0068] In this embodiment, the PCR amplification system includes: a first-round PCR amplification system, with a reaction volume of 50 μL, specifically: Mg 2+ 5–15 μL (stock solution concentration: 25 mM), 5× buffer 5–15 μL, forward and reverse primers 0.5–1.5 μL each (stock solution concentration: 20 μM), dNTP 0.5–1 μL (stock solution concentration: 100 mM), RNA nucleic acid sample 5–25 μL, reverse transcriptase 0.1–0.3 μL (stock solution concentration: 160 U / μL), Taq enzyme 0.5–1 μL (stock solution concentration: 5 U / μL), RNase inhibitor 0.3–0.7 μL (stock solution concentration: 40 U / μL), and ddH2O to bring the reaction volume to 50 μL; the reaction volume for the second round of PCR amplification is calculated as 50 μL, specifically: Mg 2+ 5–15 μL (stock solution concentration: 25 mM), 5–15 μL 5× buffer, 0.5–1.5 μL each of forward and reverse primers (stock solution concentration: 20 μM), 0.5–1 μL dNTP (stock solution concentration: 100 mM), 5–15 μL of nucleic acid product from the first round of PCR amplification, 0.5–1 μL Taq enzyme (stock solution concentration: 5 U / μL), and use ddH2O to bring the volume of the reaction system to 50 μL.

[0069] In this embodiment, the PCR amplification program includes a first-round PCR amplification program and a second-round PCR amplification program. The first-round PCR amplification program includes an RNA amplification program: reverse transcription at 50℃ for 40–50 min; pre-denaturation at 93–96℃ for 1–3 min; denaturation at 93–96℃ for 20–35 s, annealing at 45–55℃ for 25–35 s, and extension at 70–75℃ for 1–3 min, for a total of 25–40 cycles; further extension at 70–75℃ for 5–10 min, and storage at 4℃. The second-round PCR amplification program includes: pre-denaturation at 93–96℃ for 3–5 min; denaturation at 93–96℃ for 10–20 s, annealing at 50–60℃ for 15–30 s, and extension at 70–75℃ for 1–3 min, for a total of 30–45 cycles; further extension at 70–75℃ for 5–10 min, and storage at 4℃.

[0070] In this embodiment of the application, the aforementioned sequencing primer set is used during the sequencing of nested PCR amplification products.

[0071] In some preferred embodiments, the first set of upstream primers in the primer set used to amplify the HIV-1 protease and reverse transcriptase regions includes all primers with nucleotide sequences of SEQ ID NO: 1–27; the first set of downstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 28–55; the second set of upstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 56–80; the second set of downstream primers in the primer set includes all primers with nucleotide sequences of SEQ ID NO: 81–110; the selected sequencing primer set includes all 24 primers with nucleotide sequences of SEQ ID NO: 111–134; the specific process of applying the selected detection method to the detection of drug resistance mutations in the HIV-1 RNA protease and reverse transcriptase regions is as follows:

[0072] 1. Nucleic acid extraction:

[0073] This method requires first extracting RNA from serum, plasma, semen, urine, or cerebrospinal fluid, and then detecting the concentration of the extracted HIV-1 RNA nucleic acid.

[0074] (1) RNA nucleic acid extraction (13 samples)

[0075] Add 800 μL of lysis buffer to 300 μL of serum / plasma sample, vortex for 30 s, and incubate at room temperature for 10 min. Add 300 μL of isopropanol, invert and mix for 30 s, and incubate at room temperature for 2 min. Centrifuge briefly, transfer 710 μL to a centrifugal adsorption column, and centrifuge at 10000 rpm for 1 min. Discard the 2 mL collection tube, transfer the centrifugal adsorption column to a new 2 mL collection tube, and repeat twice until all samples are transferred to the centrifugal adsorption column. Discard the 2 mL collection tube, transfer the centrifugal adsorption column to a new 2 mL collection tube, add 500 μL of wash buffer 1, and centrifuge at 10000 rpm for 1 min. Discard the 2 mL collection tube, transfer the centrifugal adsorption column to a new 2 mL collection tube, add 500 μL of wash buffer 2, and centrifuge at 14000 rpm for 3 min. Discard the 2 mL collection tube, transfer the centrifugal adsorption column to a new 2 mL collection tube, and centrifuge at 14000 rpm for 1 min. Discard the 2 mL collection tube, 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.

[0076] The concentration of HIV-1 RNA was quantitatively detected using real-time quantitative PCR, and the results are shown in Table 1.

[0077] Table 1

[0078]

[0079] 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 samples with levels >1000 copies / mL have a drug resistance success rate of around 85%.

[0080] 2. First round of nucleic acid PCR amplification:

[0081] (1) Preparation of reaction system: Mg 2+10 μL (stock solution concentration: 25 mM), 10 μL 5× buffer, 1.25 μL each of forward and reverse primers (stock solution concentration: 20 μM), there are 55 primers, the nucleotide sequences of which are shown in SEQ ID NO: 1~55, 1 μL dNTP (stock solution concentration: 100 mM), 0.125 μL reverse transcriptase (stock solution concentration: 160 U / uL), 0.8 μL Taq enzyme (stock solution concentration: 5 U / uL), 0.5 μL RNase inhibitor (stock solution concentration: 40 U / uL), and 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).

[0082] (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.

[0083] (3) Add 20 μL of the extracted and purified nucleic acid sample, label it, vortex for 30 seconds, and then centrifuge quickly.

[0084] (4) PCR amplification: After inserting the eight-piece array into the instrument, select the following PCR amplification program:

[0085] Reverse transcription at 50℃ for 45 min; pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 30 cycles; extension at 72℃ for 10 min, and storage at 4℃.

[0086] The experiment lasted approximately 3 hours.

[0087] 3. Second round of nucleic acid PCR amplification:

[0088] (1) Preparation of reaction system: Mg 2+ 10 μL (stock solution concentration: 25 mM), 10 μL 5× buffer, 1.25 μL each of forward and reverse primers (stock solution concentration: 20 μM), there are 55 primers, the nucleotide sequences of which are shown in SEQ ID NO: 56~110, 1 μL dNTP (stock solution concentration: 100 mM), 0.8 μL Taq enzyme (stock solution concentration: 5 U / uL), and the volume of the reaction system was made up to 40 μL using ddH2O;

[0089] (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.

[0090] (3) Add 10 μL of the extracted and purified nucleic acid sample, label it, vortex for 30 seconds, and then centrifuge quickly.

[0091] (4) PCR amplification: After inserting the eight-piece array into the instrument, select the following PCR amplification program:

[0092] Pre-denaturation at 94℃ for 4 min, denaturation at 94℃ for 15 s, annealing at 55℃ for 20 s, extension at 72℃ for 2 min, 37 cycles; extension at 72℃ for 10 min, storage at 4℃.

[0093] The experiment lasted approximately 3 hours.

[0094] 4. Agarose gel electrophoresis analysis of PCR amplification products:

[0095] TAE dilution: 10 mL (50×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.

[0096] Agarose gel electrophoresis analysis results: such as Figure 1 As shown in Table 2, the target band is between 1000 and 2000 bp. The appearance of the band indicates successful amplification, and subsequent drug resistance testing can be performed. The test results are shown in Table 2. When the HIV-1 RNA sample concentration is greater than 500 copies / mL, the detection rate is 100%, and the success rate in all concentration ranges is much higher than that of existing technologies.

[0097] Table 2

[0098]

[0099] 5. Sequencing and analysis of PCR amplification products:

[0100] (1) Sequencing of PCR amplification products: The target band of the above PCR amplification was cut out and purified;

[0101] 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.

[0102] 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: 111-134.

[0103] (2) Post-sequencing data analysis:

[0104] 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 graph, align according to drug resistance sites, and check for gene mutations; after completion, save the project and export the assembled sequences to the specified folder; open the HIV DRUG RESISTANCE DATABASE drug resistance analysis website, analyze the mutation status of the sequences, and calculate the information of drug resistance sites and the degree of drug resistance, such as... Figure 2 As shown.

[0105] 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.

[0106] Subtype analysis:

[0107] The concatenated sequence is then used to determine subtypes in a phylogenetic tree manner, such as... Figure 3As shown, this method can currently complete 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, CRF49_CPX, CRF78_CPX, CRF96_CPX, CRF06_CPX, CRF09_CPX, CRF11_CPX, CRF13_CPX, CRF14_BG, CRF15_01B, CRF16_A2D, CRF18_CPX, CR F19_CPX, CRF20_BG, CRF04_CPX, CRF22_01A1, CRF23_BG, CRF24_BG, CRF25_CPX, CRF26_A5U, CRF27_CPX, CRF29_ BF1, CRF31_BC, CRF33_01B, CRF34_01B, CRF35_A1D, CRF36_CPX, CRF37_CPX, CRF38_BF1, CRF39_BF1, CRF40_BF 1. CRF43_02G, CRF47_BF1, CRF48_01B, CRF51_01B, CRF53_01B, CRF54_01B, CRF56_CPX, CRF57_BC, CRF58_01B, C Drug resistance testing for multiple HIV-1 subtypes, including RF59_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.

[0108] 6. Test Results and Analysis

[0109] Figure 2 The information diagram of the drug resistance site provided in the embodiments of this application is a schematic diagram of the drug resistance mutation site of HIV-1 RNA and the region where the amplification product is located. The diagram shows the mutation status of the sequence in its respective enzyme region. For example, K20R represents a mutation at amino acid number 20 in the protease region, where the amino acid changes from K to R. Similarly, the mutation status of all bases in the region can be obtained from the diagram.

[0110] Figure 3The phylogenetic tree diagram provided in this application embodiment is a phylogenetic tree diagram based on HIV-1 RNA as the sample type. The bolded parts in the diagram represent the standard sequences of different subtypes. The test samples are named with numbers, and the subtype analysis of unknown samples is performed using a phylogenetic tree model.

[0111] 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 detecting HIV-1 RNA protease and reverse transcriptase regions, 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: 1-27; the first set of downstream primers comprises all primers with nucleotide sequences of SEQ ID NO: 28-55; 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: 56-80; the second set of downstream primers includes all primers with nucleotide sequences of SEQ ID NO: 81-110.

2. The primer set for detecting HIV-1 RNA protease and reverse transcriptase regions according to claim 1, characterized in that, It also includes a sequencing primer set, which is used to sequence the amplified products of HIV-1 protease and reverse transcriptase regions, wherein the amplified products are obtained by PCR amplification using the primer set of claim 1; the sequencing primer set includes primers with nucleotide sequences of SEQ ID NO: 111-134, and the sequencing primer set includes forward sequencing primers and reverse sequencing primers.

3. A kit for detecting drug resistance site mutations in the protease and reverse transcriptase regions of HIV-1 RNA, characterized in that, The kit includes RNA extraction reagent, RNA reverse transcription reagent, and the primer set as described in claim 2.

4. A method for detecting mutations in the HIV-1 RNA protease and reverse transcriptase regions, wherein the detection method is not for disease diagnosis or treatment purposes, characterized in that, The detection method includes: RNA was extracted from the sample to be tested and 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 mutation results of the HIV-1 RNA protease and reverse transcriptase regions.

5. The detection method according to claim 4, 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.

6. The detection method according to claim 5, characterized in that, The reaction volume for the first round of PCR amplification was calculated to be 50 μL, specifically as follows: Mg 2+ 5~15 μL, 5×buffer 5~15 μL, forward and reverse primers 0.5~1.5 μL each, dNTP 0.5~1 μL, RNA nucleic acid sample 5~25 μL, reverse transcriptase 0.1~0.3 μL, Taq enzyme 0.5~1 μL, RNase inhibitor 0.3~0.7 μL, and use ddH2O to make up the volume of the reaction system to 50 μL; The reaction volume for the second round of PCR amplification is calculated to be 50 μL, specifically as follows: Mg 2+ 5~15 μL, 5×buffer 5~15 μL, forward and reverse primers 0.5~1.5 μL each, dNTP 0.5~1 μL, nucleic acid product from the first round of PCR amplification 5~15 μL, Taq enzyme 0.5~1 μL, and use ddH2O to make up the volume of the reaction system to 50 μL.

7. The detection method according to claim 5, characterized in that, The procedure for the first round of PCR amplification includes: Reverse transcription at 50 ℃ for 40-50 min; pre-denaturation at 93-96 ℃ for 1-3 min; denaturation at 93-96 ℃ for 20-35 s, annealing at 45-55 ℃ for 25-35 s, extension at 70-75 ℃ for 1-3 min, for a total of 25-40 cycles; further extension at 70-75 ℃ for 5-10 min, and storage at 4 ℃; The procedure for the second round of PCR amplification includes: Pre-denaturation at 93~96℃ for 3~5 min; denaturation at 93~96℃ for 10~20 s, annealing at 50~60℃ for 15~30 s, extension at 70~75℃ for 1~3 min, for a total of 30~45 cycles; further extension at 70~75℃ for 5~10 min, and storage at 4℃.

Images