Primer group for detecting HIV-1 genome, kit containing primer group and application

Abstract

The invention discloses a primer group for detecting an HIV-1 (human immunodeficiency virus-1) genome, a kit containing the primer group and application. The primer group comprises nucleotide sequences as shown in SEQ ID NO.1-65. The primer group can be used for amplifying a plurality of subtype or recombinant HIV-1 type genomes. The invention also provides a method for detecting an HIV-1 type genome, which comprises the following steps: firstly, carrying out reverse transcription on the HIV-1 type genome by virtue of an efficient reverse transcription system with high thermal stability and enhanced affinity with a template, and efficiently synthesizing cDNA (complementary deoxyribonucleic acid); then amplifying the HIV-1 type cDNA by using a high-specificity primer pool and a high-fidelity enzyme which are mixed in advance by adopting a multiple PCR (Polymerase Chain Reaction) amplification principle, so as to obtain an HIV-1 type genome sequence in the sample; and finally, the detection of the HIV-1 type genome is realized through a nanopore three-generation sequencing platform. According to the detection method, nucleotide mutation can be analyzed through sequence alignment, the method has the characteristics of simplicity and convenience in operation, high efficiency, high specificity, accuracy and rapidness, and a brand new method is provided for identifying HIV-1 type drug-resistant mutation types.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, specifically to a primer set for detecting the HIV-1 genome, a kit containing the primer set, and its application. Background Technology

[0002] AIDS, or Acquired Immunodeficiency Syndrome, is caused by infection with the Human Immunodeficiency Virus (HIV), a progressive immune deficiency characterized by a decrease in CD4+ T lymphocytes. In later stages, it can lead to various opportunistic infections, malignancies, and central nervous system disorders. It is the leading cause of death from infectious diseases, particularly in southern African countries. In 2023, 630,000 people worldwide were still dying from HIV, and 39.9 million were still living with the virus.

[0003] Antiretroviral therapy (ART) is currently the core strategy for treating HIV / AIDS. It inhibits HIV replication by disrupting key steps in the HIV-1 replication cycle through multiple targets, thereby slowing the virus's damage to the immune system and prolonging the patient's lifespan. Since zidovudine was approved in 1987, more than 30 antiretroviral drugs have been developed. The life expectancy of HIV-infected individuals receiving antiretroviral therapy can approach that of the general population. However, due to the rapid replication of HIV and its lack of self-correction during replication, it is prone to gene mutations leading to drug resistance. Furthermore, under the selective pressure of long-term antiretroviral drugs, the probability of HIV drug resistance mutations increases significantly. In 2019, approximately 67% of HIV-infected individuals worldwide received ART. Due to the increased use of ART, the prevalence of drug resistance mutations (DRM) is also rising, posing a significant obstacle to achieving the UN HIV / AIDS program's goal of eliminating HIV by 2030.

[0004] Currently, the main methods for detecting drug resistance are genotypic and phenotypic testing. Genotypic drug resistance testing uses polymerase chain reaction (PCR) to amplify the protease, reverse transcriptase, and even integrase genes of HIV / AIDS samples, and then performs sequence determination and analysis of the amplified products to determine the presence of drug-resistant variants. The advantages of this method are its simplicity and low cost; the results can be interpreted based on established variant models related to phenotypic drug resistance. However, the detection is limited to dominant strains and requires expert interpretation. Phenotypic testing requires viral isolation and culture of HIV / AIDS samples to prepare high-titer HIV strains, followed by drug susceptibility testing. This method is the gold standard for HIV-1 drug resistance detection. Its advantage is that the results are easily interpreted, but the testing cost is very high, and the time required is long, taking 4-6 weeks to obtain results. This method is also limited to detecting major drug-resistant strains.

[0005] According to UNAIDS's concept of "undetectable = non-transmittable," the World Health Organization's (WHO) recommended universal testing and treatment strategies, as well as expanding pre-exposure prophylaxis among high-risk individuals, are consensus strategies for reducing the global spread of HIV. However, sequencing data using genotypic resistance testing and molecular epidemiology-based surveillance can also help identify expanding HIV transmission clusters and implement targeted prevention measures.

[0006] Commercially available detection methods have limitations: they can only detect a limited number of target regions and drug-resistant gene mutations, and they cannot distinguish specific mutation types. Therefore, considering current market demands, the development of a rapid and efficient detection kit for the human immunodeficiency virus type 1 genome is urgently needed. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide a primer set for detecting the HIV-1 genome, a kit containing the primer set, and an application thereof. Based on the target region of the HIV-1 genome, a primer set that can distinguish various drug resistance gene mutation types is provided, and a new generation nanopore sequencing method for HIV-1 gene drug resistance mutations is constructed.

[0008] To achieve the above objectives, this application provides at least the following technical solutions: In a first aspect, this application provides a primer set for detecting the HIV-1 gene, comprising the nucleotide sequence shown in SEQ ID NO.1-65.

[0009] By adopting the above technical solution, this application designs a set of multiplex PCR primers for conserved regions of HIV-1 to fragment the target gene, facilitating sequencing analysis of its drug resistance mutations. Through sequence alignment, nucleotide mutations in the HIV-1 gene can be analyzed. The primer set has high sensitivity, high accuracy, and good reproducibility, and can amplify the genomes of multiple subtypes or recombinant HIV-1 types, reporting specific mutation types and mutation ratios, and can more effectively detect HIV-1 prevalent strains in China.

[0010] Preferably, the HIV-1 gene includes one or more of the CRF01_AE subtype, CRF07_BC subtype, CRF08_BC subtype, and B' subtype.

[0011] By employing the above-mentioned technical solution, this application designed a primer set by comparing the gene sequences of CRF01_AE, CRF07_BC, CRF08_BC, and B' subtypes. This primer set is used for multiplex PCR amplification of target genes, thereby constructing a nanopore sequencing system to achieve high-throughput, high-sensitivity, and high-specificity screening for HIV-1 drug resistance mutations. The detection range covers one or more novel and unique recombinant types from CRF01_AE, CRF07_BC, CRF08_BC, and B' subtypes, offering broader coverage and better compatibility, which is beneficial for epidemiological research or clinical auxiliary diagnosis. Through extensive sequence alignment and practical application, the primers obtained in this application have shown good specificity and sensitivity for the relatively prevalent HIV-1 strain in China.

[0012] Secondly, this application provides a kit for detecting HIV-1 gene drug resistance mutations, including a primer set for detecting the HIV-1 gene.

[0013] Thirdly, this application provides a screening method for detecting HIV-1 genotypes or drug resistance mutations for purposes other than disease diagnosis and / or treatment, comprising the following steps: (1) Design multiplex PCR primers for the HIV-1 genome and mix them into a primer pool; (2) Use the primer pool in (1) to perform PCR amplification on the HIV-1 nucleic acid sample to be tested, and then purify it; (3) The amplification product obtained in (2) was repaired at the end so that its 5' end contained a phosphate group and its 3' end had a dA protrusion, and then purified. (4) The product obtained in (3) is subjected to dT protrusion and barcode linkage, and then purified; (5) The product obtained in (4) is ligated to the nanopore sequencing adapter, purified, and the final HIV-1 gene library is obtained. (6) Sequencing was performed using a nano-sequencer. After quality control analysis of the gene library obtained in (5), data analysis was conducted.

[0014] In some implementations, in step (1), the multiplex PCR primers are the primer set described in the first aspect.

[0015] In some implementations, step (2) of the PCR amplification reverse transcription includes two steps: denaturation and reverse transcription.

[0016] In some implementations, in step (2), the PCR amplification system is: 3µL of cDNA, 15µL of premixed solution, 3µL of primer pool, and water added to 30µL. The amplification conditions were as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60℃ annealing for 2.5 min, 72℃ extension for 45 s, for 35 cycles; after the cycles, 72℃ extension for 5 min, and storage at 4℃. The product was purified using DNA purification magnetic beads.

[0017] In some implementations, in step (3), the end-repair system comprises: 15 µL of EP Mix, 200 ng of PCR purified product, and water to a final volume of 65 µL; The end-repair conditions were: reaction at 20℃ for 15 min, reaction terminated at 65℃ for 15 min, and stored at 4℃. The product was purified using DNA purification magnetic beads.

[0018] In some implementations, in step (4), the ligation system of the barcode includes: 12µL of unrefined purified product, 5µL of fast DNA ligase, 7µL of fast ligation buffer and 1µL of sequencing ligation adapter. The connection conditions are: react at 20℃ for 20 min, and store at 4℃.

[0019] In some embodiments, the purification is performed using DNA purification magnetic beads to purify the product.

[0020] In some implementations, in step (5), the sequencing adapter ligation system is: 25 µL of the purified barcode ligation product, 10 µL of DNA ligase, 12.5 µL of ligation buffer, and 2.5 µL of sequencing ligation adapter. The sequencing ligation procedure is as follows: react at 20°C for 30 min, then store at 4°C; Product purification was performed using DNA purification magnetic beads.

[0021] In some specific implementations, step (6) includes: (1) Preparation of inducing agent: 20µL of sequencing chip tether and 780µL of sequencing chip washing solution; (2) Open the induction well of the sequencing chip, add 550 µL of inducing agent, let stand for 5 min, and then add another 200 µL of inducing agent; (3) Preparation of reaction solution for sequencing: 30µL sequencing buffer, 15µL library particles and 15µL adapter ligation and purification library; (4) Open the sample well of the sequencing chip, add 60 µL of the sequencing reaction solution and close all the well caps. Set the sequencing program and start sequencing.

[0022] After filtering out invalid data such as connectors and performing quality control analysis on the data from step (6) using the above method, the sequence is compared with the HIV-1 genome to determine the type of gene mutation in the test sample.

[0023] Fourthly, this application provides an application of a primer set in detecting HIV-1 gene drug resistance mutations.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. The specific primer set designed in this application can be used to amplify common subtypes of HIV-1 strains in clinical practice. A wide variety of anti-HIV drugs are available, which can help clinicians guide patients in medication use and provide a reference. 2. This application utilizes multiplex PCR combined with nanopore sequencing technology to sequence the human HIV-1 genome, overcoming the technical limitations of current commercially available HIV-1 detection methods, such as limited applicability to subtypes and low throughput. Furthermore, it allows for more comprehensive and accurate identification of human HIV-1 types, mitigating false negatives and false positives caused by insufficient single-gene identification capabilities. 3. The detection method of this application can detect both subtypes and human HIV-1 drug resistance, with a short detection cycle, high sensitivity, and good specificity. Attached Figure Description

[0025] Figure 1 This is a fluorescent PCR result image of HIV-1 identification for samples H1-H25 in Example 3 of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0027] The materials used in the following embodiments are not limited to those listed above and may be replaced by other similar materials. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0029] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0030] In the context of this application, many embodiments use the terms "comprising" or "including". The terms "comprising" or "including" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, and method steps specifically listed after the statement, but also other elements, components, parts, and method steps. However, in this document, the terms "comprising" or "including" can also be understood as closed-ended expressions in certain cases, indicating that they only include the elements, components, parts, and method steps specifically listed after the statement, and exclude any other elements, components, parts, or method steps. In this case, the statement is equivalent to the statement "composed of...".

[0031] Example 1: Primer Design The genome sequences of the most prevalent HIV-1 strains currently circulating in my country were downloaded from the HIV database. These subtypes include CRF01_AE, CRF07_BC recombinant, CRF08_BC, and subtype B'. Sequence alignment was performed using DNAman software. Based on the sequence analysis results, conserved regions were selected, and primers capable of amplifying the entire genome sequence were designed. Primer sequence information is shown in Table 1.

[0032] Table 1. Primer sequences for multiplex PCR

[0033] In the primer sequences above, A represents adenine, G represents guanine, C represents cytosine, T represents thymine, the degenerate base R represents A / G, Y represents C / T, M represents A / C, K represents G / T, W represents A / T, and H represents A / C / T.

[0034] Example 2: Feasibility Verification of the Procedure for Detecting HIV-1 Genome Based on Nanopore Sequencing 1. Nucleic acid extraction from clinical samples The body fluid nucleic acid extraction kit was purchased from Zhejiang Shengting Biotechnology Co., Ltd. Sequencing was performed using the MiniIon and GridIon nanopore platforms from Nanopore. The sequencing kit was SQK-NBD114, and the sequencing chip was R10. The specific steps are as follows: (1) Take 250 µL of each of the seven samples (numbered A1, A2, A3, A4, A5, A6, A7) and the negative control, and add 5 µL of lysozyme, 10 µL of cell wall lysin, and 20 µL of proteinase K. Incubate in a metal bath at 30 °C for 15 min, add 400 µL of lysis binding buffer, mix well, and incubate at 70 °C for 5 min. After incubation, briefly separate the sample, take 400 µL of isopropanol into an EP tube, add 20 µL of mixed magnetic beads into the sample, vortex for 10-15 s, and let stand at room temperature for 5 min. Transfer the sample to a magnetic rack for adsorption for 1 min, and discard the supernatant. (2) Washing: Add 600µL of washing solution 1, vortex mix for 30 s, resuspend the magnetic beads, transfer the sample to the magnetic rack for adsorption for 30 s, and discard the supernatant; add 600µL of washing solution 2, vortex mix for 30 s, resuspend the magnetic beads, transfer the sample to the magnetic rack for adsorption for 30 s, and discard all residual supernatant; add 600µL of washing solution 2 again, vortex mix for 30 s, resuspend the magnetic beads, transfer the sample to the magnetic rack for adsorption for 30 s, and discard all residual supernatant; (3) Elution: Open the tube cap and dry at room temperature for 5-15 min (until matte); add 100 µL of elution buffer, vortex to mix, place at 70℃ and incubate for 3 min; transfer the sample tube to a magnetic rack for 1 min to adsorb, transfer the supernatant to a new 1.5 mL centrifuge tube and store at -20℃.

[0035] 2. Reverse transcription of nucleic acids The extracted nucleic acid was reverse transcribed in a 0.2 ml PCR tube. The reverse transcription system consisted of 2 µL RT Mix, 2 µL RTEnzyme Mix, 1 µL RT Primer, 10 µL nucleic acid, and 5 µL RNase-free H2O. The tube was gently tapped to mix, briefly centrifuged, and then placed in a PCR amplification instrument for reverse transcription to obtain cDNA. The reverse transcription parameters were adjusted to 37°C for 30 min and 85°C for 5 s for inactivation.

[0036] 3. PCR amplification and enrichment of genomic DNA and product purification (1) PCR amplification: Prepare the PCR amplification system according to the following formula: Input cDNA 3µL, PCR Mix 15µL, primer pool 3µL, nuclease-free water 9µL. Close the tube cap, vortex to mix, and briefly incubate on a PCR instrument for multiplex PCR amplification. The multiplex PCR amplification reaction conditions are: 95℃ pre-denaturation for 2 min, 95℃ pre-denaturation for 30 s, 60℃ annealing for 2.5 min, 72℃ extension for 45 s, for 35 cycles; 72℃ extension for 5 min, and store at 4℃. (2) PCR product purification: ① Remove the purification magnetic beads in advance and equilibrate at room temperature for at least 30 min, then vortex to mix. ② After PCR, remove the sample tubes and briefly centrifuge. ③ Add 30 µL of purification magnetic beads to each sample tube, vortex or gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min. ④ Briefly centrifuge the PCR tubes and place them in a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant. ⑤ Keep the PCR tubes in the magnetic rack at all times, add 200 µL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. ⑥ Repeat step ⑤, rinsing twice in total. ⑦ Keep the PCR tubes in the magnetic rack at all times, open the caps and air dry the magnetic beads for 5-10 min until no ethanol residue remains. ⑧ Remove the PCR tubes from the magnetic rack, add 30 µL of enzyme-free water to each tube, gently vortex or gently pipette to mix thoroughly, and place at room temperature for 5 min. min; ⑨ Briefly centrifuge the PCR tube and place it on a magnetic rack to stand until the solution becomes clear (about 5 min). Carefully transfer 28 µL of supernatant to a new 1.5 mL centrifuge tube, being careful not to touch the magnetic beads; ⑩ Determine the concentration of the purified product using the Qubit™ fluorometer according to the Qubit instructions. The concentrations of the purified products are: A1: 112.1 ng / µL, A2: 89.8 ng / µL, A3: 90.2 ng / µL, A4: 9.8 ng / µL, A5: 108 ng / µL, A6: 7.7 ng / µL, A7: 63.1 ng / µL, NC (negative control): 1.3 ng / µL.

[0037] 4. End-of-phase repair and product purification Thaw the EP Mix and invert it to mix thoroughly. Perform end-repair in a sterile PCR tube. The end-repair reaction mixture consists of 500 ng of multiplex PCR purified product, 15 µL of EP Mix, and 50-X nuclease-free water, totaling 65 µL. Gently pipette to mix (do not vortex), and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a PCR instrument for the reaction under the following conditions: heat-cap at 105°C, react at 20°C for 15 min, terminate the reaction at 65°C for 15 min, and store at 4°C. Purification of end-repair products: ① Remove the purification magnetic beads in advance and equilibrate at room temperature for at least 30 min, then vortex to mix. ② After end-repair, remove the sample tubes and centrifuge briefly. ③ Add 65 µL of purification magnetic beads to each sample tube, vortex or gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min. ④ Briefly centrifuge the PCR tubes and place them on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant. ⑤ Keep the PCR tubes on the magnetic rack at all times, add 200 µL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. ⑥ Repeat step ⑤, rinsing twice in total. ⑦ Keep the PCR tubes on the magnetic rack at all times, open the caps and air dry the magnetic beads for 5-10 min until no ethanol residue remains. ⑧ Remove the PCR tubes from the magnetic rack, add 15µL of enzyme-free water to each tube, gently vortex or pipette to mix thoroughly, and let stand at room temperature for 2 min; ⑨ Briefly centrifuge the PCR tubes and place them on the magnetic rack to stand until the solution becomes clear (about 5 min). The supernatant is the purified library after end-repair.

[0038] 5. Amplification-free barcode connection (1) Thaw 5µL of rapid DNA ligase, 7µL of rapid ligation buffer, NB X, and EDTA, then invert and mix well, and place on ice. (2) Based on the number of samples, thaw the corresponding number of amplification-free NB X (NB01-24) at room temperature. Mix them thoroughly by pipetting, then centrifuge briefly and place on ice; (3) In a sterile PCR tube, the barcode ligation reaction system is as follows: 12µL of unpurified product, 5µL of Rapid DNA Ligase, 7µL of Rapid Ligation Buffer, and 1µL of NB X (SQK-NBD114); (4) Use a pipette to gently mix the liquid by blowing (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube; (5) Place the PCR tube in the PCR instrument. The barcode connection reaction conditions are: 105℃ hot cap, 20℃ reaction for 20 min, and 4℃ storage. (6) After the PCR reaction is completed, centrifuge briefly, add 5µL of EDTA (blue cap) to the above ligation reaction system, mix well, and centrifuge briefly; (7) Purification of barcode ligation products: ① Mix the samples ligated with different barcodes in a 1.5 mL centrifuge tube, and mix the DNA samples with ligated barcodes; ② Remove the purification magnetic beads in advance, equilibrate at room temperature for at least 30 min, and vortex to mix; ③ Add 0.8x purification magnetic beads to each sample tube, vortex or gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5-10 min; ④ Briefly centrifuge the PCR tube and place it in a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant; ⑤ Keep the PCR tube in the magnetic rack at all times, add 700 µL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant; ⑥ Repeat step ⑤, rinsing twice in total; ⑦ Keep the PCR tube in the magnetic rack at all times, open the cap and air dry the magnetic beads until there is no ethanol residue and the surface is not cracked; ⑧ Remove the PCR tube from the magnetic rack and add 30 µL of freshly prepared 80% ethanol to each tube. Mix µL of enzyme-free water thoroughly by gently pipetting; ⑨ Incubate at 37 ℃ for 10 minutes. Gently tap the centrifuge tube for 10 seconds every two minutes to agitate the sample and promote DNA elution; ⑪ Briefly centrifuge the PCR tube and place it on a magnetic rack to stand until the solution becomes clear (about 5 minutes). The supernatant is the purified library of the barcode ligation product; ⑫ Determine the concentration of the purified product using the Qubit™ fluorometer according to the Qubit instructions; the concentration of the purified product is 40.8 ng / µL.

[0039] 6. Sequencing adapter connection (1) After thawing DNA ligase, ligation buffer, and NA (SQK-NBD114), mix them by inversion and place them in a sterile PCR tube. The sequencing adapter ligation reaction system is as follows: 25µL of barcode purified product, 10µL of DNA ligase, 12.5µL of ligation buffer, and 2.5µL of NA (SQK-NBD114). (2) Gently blow on the mixture to mix it thoroughly, and then centrifuge it briefly. (3) Place the PCR tube in the PCR instrument. The sequencing adapter connection reaction is as follows: heat the cap at 105℃, react at 20℃ for 20 min, and store at 4℃. (4) Sequencing adapter ligation and purification: ① Remove the purification magnetic beads from the 4 ℃ freezer in advance and equilibrate at room temperature for at least 30 min, then vortex to mix; ② After adapter ligation, remove the sample tubes and briefly centrifuge; ③ Add 30 µL (0.6x) purification magnetic beads to each sample tube, vortex or gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5-10 min; ④ Briefly centrifuge the PCR tubes and place them on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant; ⑤ Keep the PCR tubes on the magnetic rack at all times, add 125 µL SFB to rinse the magnetic beads, gently tap the centrifuge tube to mix the magnetic beads, briefly centrifuge, place on the magnetic rack, and carefully remove the supernatant after the magnetic beads are completely adsorbed; ⑥ Repeat step ⑤, rinsing twice in total; ⑦ Keep the PCR tubes on the magnetic rack at all times, and use a 10 µL pipette tip to remove any remaining liquid; ⑧ Remove the PCR tubes from the magnetic rack and add 15 µL to each tube. Gently mix the TE elution buffer by pipetting; incubate at 37°C for 10 minutes; gently tap the centrifuge tube for 10 seconds every two minutes to agitate the sample and promote DNA elution; briefly centrifuge the PCR tube and place it on a magnetic rack to stand until the solution becomes clear (approximately 5 minutes). The supernatant is the purified library containing the adapter; determine the concentration of the purified product using the Qubit™ fluorometer according to the Qubit instructions. The concentration of the purified product is 32.8 ng / µL.

[0040] 7. Document Library Computer Lab Thaw the sequencing buffer (SB), library particles (LIB), sequencing chip tether (FCT), and one tube of sequencing chip wash buffer (FCF) from the Nanopore SQK-NBD114 kit at room temperature. After complete thawing, vortex to mix, then briefly centrifuge and place on ice.

[0041] (1) Reagent preparation: ①Inducing agent: Take a new 1.5 mL centrifuge tube and add 20 µL of FCT and 780 µL of FCF; ②Reaction solution: Take a new 1.5 mL centrifuge tube and add 30 µL of SB, 15 µL of LIB, 200 ng of the purified library after adapter ligation and nuclease-free water 60-X, and add water to 60 µL.

[0042] (2) Onboarding: ① Open the well for adding the inducing reagent, adjust the 1000µL pipette to a small range, align the pipette tip with the inducing well, adjust the range to a large range, and aspirate a small amount of liquid and discard it (ensure no air enters the chip); ② Replace with a new pipette tip, aspirate 550µL of inducing reagent, adjust the range slightly before adding the inducing reagent so that the pipette tip has a small liquid droplet, insert it into the well, adjust the range to a small range so that the liquid slowly enters the chip, until the range cannot be adjusted to a small range; ③ After the inducing reagent is added, let it stand for 5 minutes; ④ Open the sample well (SpotOnsample port), and continue to add 200µL of inducing reagent according to the previous step.

[0043] (3) Sample loading: ① Use a 100 µL pipette to aspirate 60 µL of the library for sequencing (mix well by pipetting before aspirating); ② When loading the sample, hold the pipette tip above the sample well and slowly press the pipette to ensure that the library is added drop by drop; ③ After loading the sample, cover the sample well with the cap, close the induction well, insert the selected sequencing channel, and cover with the light shield; ④ Set the sequencing program and start sequencing.

[0044] (4) Data offload quality control: After the data is offloaded, quality control analysis is performed on the offloaded data: sequences with a length of less than 250 bp are filtered to obtain the target sequence. Then the sequence is compared with the human immunodeficiency virus type 1 genome sequence in the database.

[0045] (5) Data Analysis If the quality-controlled sequence matches the HIV-1 genome sequence in the database, it can be determined that the experiment detected the corresponding subtype of HIV-1.

[0046] The results showed that CRF01_AE strain was detected in sample A1, CRF01_AE strain was detected in sample A2, subtype B' was detected in sample A3, HIV-1 was not detected in sample A4, CRF07_BC was detected in sample A5, HIV-1 was not detected in sample A6, subtype B' was detected in sample A7, and HIV-1 was not detected in the negative control.

[0047] It is understood that the method for detecting the full-length genome of HIV-1 in this application can be used for diagnostic purposes or for "non-diagnostic purposes". The "non-diagnostic purposes" include, but are not limited to, HIV-1 virus-related research such as drug resistance analysis, minor variation analysis and phylogenetic tree analysis, and inspection and quarantine.

[0048] Example 3: Establishment of a Multiple System The primers from Example 1 were mixed to form a primer pool. Following Example 2, HIV-1 positive samples were used to verify and analyze the amplification effect of each region and the coverage of the HIV-1 genome. The amplification effect of the primer pool was verified, and the results are shown in Table 2.

[0049] Table 2. Expanded Coverage in Each Region

[0050] As shown in Table 2, the primer pool of this application has a wide coverage area, covering key regions of the HIV-1 genome, and the coverage is uniform, making it suitable for multiplex PCR systems.

[0051] Example 4: Specificity Verification To verify the accuracy of this application in detecting HIV-1 and its specificity in detecting HIV-1-free negative samples, this embodiment tested 25 clinical samples using the detection method described in this application, and analyzed the number of reads aligned to the HIV-1 genome. The specific results are shown in Table 3. Simultaneously, the HIV-1 identification results in this application were verified using fluorescent PCR, and the results are shown in [Table 3]. Figure 1 .

[0052] Table 3 Clinical Sample Test Results

[0053] As shown in Table 3, the primer set and detection method provided in this application have good specificity for detecting HIV-1.

[0054] Depend on Figure 1 As can be seen, 14 samples showed amplification curves, which are consistent with the results in Table 2, further verifying the high specificity and accuracy of the method for detecting the HIV-1 gene provided in the embodiments of this application.

[0055] Example 5: Validation of the accuracy of drug resistance gene detection This embodiment collected 15 clinically diagnosed HIV-1 positive samples with known drug resistance types and performed testing according to the steps in Example 2. The detection results of the genotype drug resistance of the 15 clinical samples according to the method of this application are shown in Tables 4 and 5.

[0056] Table 4. Data from the machine shutdown process

[0057] Table 5 Results of drug resistance site detection

[0058] As shown in Tables 4 and 5, the above test results are consistent with the clinical diagnosis results, indicating that this application can accurately and effectively detect HIV-1 drug resistance mutation sites.

[0059] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A primer set for detecting HIV-1 gene, characterized by, comprising a nucleotide sequence as shown in SEQ ID NO. 1-65.

2. The primer set according to claim 1, characterized in that, The HIV-1 gene comprises one or more subtypes of CRF01_AE, CRF07_BC, CRF08_BC and B' subtypes of HIV-1 gene.

3. A kit for detecting HIV-1 genetic drug resistance mutations, characterized in that, The primer set of any one of claims 1-2.

4. A screening method for detecting HIV-1 genotypes or drug resistance mutations for the purpose of non-disease diagnosis and / or treatment, characterized by, comprising the following steps: (1) Designing multiplex PCR primers for HIV-1 genome, and mixing into a primer pool; (2) Using the primer pool in (1) to perform PCR amplification on the HIV-1 nucleic acid sample to be detected, and purifying; (3) Performing end repair on the amplification product obtained in (2) to make the 5' end contain a phosphate group and the 3' end have a dA overhang, and purifying; (4) Connecting dT overhang and Barcode to the product obtained in (3), and purifying; (5) Connecting nanopore sequencing adapter to the product obtained in (4), purifying, and obtaining the final HIV-1 gene library; (6) Sequencing using a nanopore sequencer, and performing data analysis after quality control analysis of the gene library obtained in (5).

5. The screening method of claim 4, wherein, The multiplex PCR primers are the primer set of claim 1.

6. The screening method of claim 4, wherein, In step (2), the PCR amplification system comprises 3 µL of cDNA, 15 µL of premix, and 3 µL of primer pool. The amplification conditions are: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s, 60°C annealing for 2.5 min, 72°C extension for 45 s, 35 cycles; after the cycles, 72°C extension for 5 min, and 4°C storage; The product purification is performed using DNA purification magnetic beads.

7. The screening method of claim 4, wherein, In step (3), the end repair system comprises 15 µL of EP Mix and 200 ng of PCR purified product. The end repair conditions are: 20°C reaction for 15 min, 65°C termination reaction for 15 min, and 4°C storage; The product purification is performed using DNA purification magnetic beads.

8. The screening method of claim 4, wherein, In step (4), the Barcode connection system comprises 12 µL of end repair purified product, 5 µL of rapid DNA ligase, 7 µL of rapid ligation buffer, and 1 µL of sequencing ligation adapter. The connection conditions are: 20°C reaction for 20 min, and 4°C storage.

9. The screening method of claim 4, wherein, In step (5), the sequencing adapter connection system is: 25 µL of Barcode connection purified product, 10 µL of DNA ligase, 12.5 µL of ligation buffer, and 2.5 µL of sequencing ligation adapter. The sequencing connection conditions are: 20°C reaction for 30 min, and 4°C storage; The purification is product purification using DNA purification magnetic beads.

10. Use of the primer set of any one of claims 1-2 in detecting HIV-1 gene drug resistance mutations.

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