CN114703178A

CN114703178A - Primer group for detecting HIV-1 integrase gene mutation and application thereof

Info

Publication number: CN114703178A
Application number: CN202210476454.0A
Authority: CN
Inventors: 廖玲洁; 邢辉; 冯毅; 宋畅; 马鹏飞; 杨晓震; 郜培杰; 史亚伦; 邵一鸣
Original assignee: Beijing Anpu Biochemical Technology Co ltd; NATIONAL CENTER FOR AIDS/STD CONTROL AND PREVENTION CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION
Current assignee: Beijing Anpu Biochemical Technology Co ltd; NATIONAL CENTER FOR AIDS/STD CONTROL AND PREVENTION CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-07-05

Abstract

The invention belongs to the technical field of biology, and provides a primer group for detecting gene mutation of an HIV-1 integrase, which comprises a first group of primer combination and a second group of primer combination; the first set of primer combination and the second set of primer combination comprise a first round amplification primer, a second round amplification primer and a sequencing primer. The invention also provides application of the primer group for detecting HIV-1 integrase gene mutation in detecting HIV-1 integrase gene mutation. The primer group for detecting the gene mutation of the HIV-1 integrase can be used for detecting samples with the viral load of more than 1000 copies/mL, simultaneously covers related drug-resistant sites of the integrase in a Stanford drug-resistant database, has high accuracy, good amplification sensitivity, high accuracy, high repeatability and lower cost when detecting the gene mutation of the integrase, is very suitable for popularization and use, and has important significance for preventing and treating AIDS.

Description

Primer group for detecting HIV-1 integrase gene mutation and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer group for detecting HIV-1 virus integrase gene mutation and application thereof.

Background

Human immunodeficiency virus (HIV-1), also known as the AIDS virus, is the causative agent of AIDS in humans. After infecting human body, the human immunodeficiency virus selectively attacks the most important CD 4T lymphocyte in human immune system and largely destroys the cell, and finally seriously destroys the normal immune function of human body. In the late stage of HIV-1 infection, HIV infection causes various opportunistic infections and tumors, and these complications affect the healthy life of people and may even be life-threatening. Data published by the united states aids planning agency (undess) in 2021 in 2020, there are about 3770 million people with existing aids virus infections worldwide, of which about 1020 million people are untreated, about 150 million people are new infected people in the same year, and about 68 million people die of aids in the same year. According to the data of the national health commission of China, as soon as 10 months in 2021, 104.5 thousands of existing AIDS infectors reported in China still have low epidemic situation, but because the spreading influence factors are more complex, the prevention situation is still severe, and the method becomes an important public health problem seriously threatening the public health of China.

After viral infection, the antiviral treatment can reduce the morbidity and the fatality rate of HIV-1 infection, the morbidity and the fatality rate of non-AIDS related diseases, ensure that a patient obtains normal expected life, improve the life quality, inhibit virus replication to the maximum extent, reduce the virus load to the lower detection limit, reduce virus variation, rebuild or improve the immune function, reduce abnormal immune activation, reduce the transmission of HIV-1 and prevent the transmission of mother and infant.

There are five major groups of anti-retroviral therapeutic drugs in China, namely Nucleoside Reverse Transcriptase Inhibitors (NRTIs), non-Nucleoside Reverse Transcriptase Inhibitors (non-NRTIs, NNRTIs), Protease Inhibitors (Protease Inhibitors, PIs), Integrase Strand and Transfer Inhibitors (INSTIs), and membrane Fusion Inhibitors (Fusion Inhibitors, FIs). According to the Chinese AIDS diagnosis and treatment guideline 2021, the currently recommended first-line treatment scheme is that two NRTIs backbone drugs are combined with a third drug, the third drug can be NNRTIs or enhanced PIs (containing ritonavir or cobicistat) or INSTIs, and the conditional patients can adopt compound single-tablet preparations.

Two HIV-1 drug resistance detection methods are currently approved by the FDA in the United states, Yapei ViroSeq^TMGenotype drug resistance test system and genotyping test for Euonymus diagnostics (Vela diagnostics) for HIV-1 genotyping. Chinese approved two genotype drug resistance detection methods, Yapei ViroSeq^TMA genotype drug resistance detection system (registration card number: national institute of mechanical education 20173405224) and a Daan gene (HIV-1) drug resistance genotype detection (registration card number: national institute of mechanical education 20153401697). Among the several products that have been certified for registration, the product detection principle of yapei and dada genes is similar, and is used for qualitative detection of drug-resistant mutations in the human immunodeficiency virus type 1 (HIV-1) genome Protease (PR) region and the Reverse Transcriptase (RT) region in clinical plasma samples. Genotypic drug resistance testing for Eulera diagnostics simultaneously detects drug-resistant mutations IN the protease, reverse transcriptase and Integrase (IN) regions. No in vitro diagnosis and detection product for detecting the drug-resistant mutation of the integrase exists in China.

Since 2009, the national drug administration has approved integrase drugs or integrase-containing complex preparations such as latilazir, dolastavir, seimerkia, jiefukang, and bitorubin. Although China has not used the medicines in a large scale, the medicines are listed as first-line antiviral treatment medicines in Chinese AIDS diagnosis and treatment guidelines (2021 edition), and Ecohniki tablets (trade name: Jifukang) lamivudine dolabrawei tablets (trade name: Duoweitong) and Bikeenalano tablets (trade name: Bituoweiwei) are brought into medical insurance after medical insurance negotiation, so that understanding of mutation conditions of integrase has an important influence on guiding clinical medication, and a corresponding mutation detection method is needed to meet the clinical requirement of integrase gene detection.

Disclosure of Invention

In order to solve the problems in the prior art, the inventor of the present invention selects a conserved site of the pol region integrase region in the HIV-1 genome database accumulated in china for many years, designs a conserved region degenerate primer, amplifies the viral RNA in the plasma of HIV-1 infected persons, obtains the gene sequence of the viral integrase region by a one-generation gene sequencing method, compares the gene sequence with a standard viral strain in a Stanford database to obtain the mutation condition of the viral integrase region, analyzes the result of whether the virus is resistant to drugs, and further obtains the present invention.

The first aspect of the present invention provides a primer set for detecting mutations in the gene of HIV-1 integrase, said primer set comprising a first set of primer combinations and a second set of primer combinations;

the first set of primer combinations comprises a first round amplification primer, a second round amplification primer and a sequencing primer:

the first round amplification primers comprise:

forward primer 1: 5 '-GGRATYATTCARGCACAACCAG-3' (SEQ ID NO: 1 in the sequence Listing);

forward primer 2: 5 '-GCATTAGGRATYATTCARGCAC-3' (SEQ ID NO: 2 in the sequence Listing);

reverse primer: 5 '-TGGGATRTGTACTTCYGARCTTA-3' (SEQ ID NO: 3 in the sequence Listing);

the second round amplification primers comprise:

a forward primer: 5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer: 5 '-CATCCTGTCTACYTGCCACAC-3' (SEQ ID NO: 5 in the sequence Listing);

the sequencing primer comprises:

forward primer 1: 5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer 1: 5 '-CATCCTGTCTACYTGCCACAC-3' (SEQ ID NO: 5 in the sequence Listing); forward primer 2: 5 '-GGVATTCCCTACAATCCCCAAAG-3' (SEQ ID NO: 6 in the sequence Listing);

and a reverse primer 2: 5'-GAATACTGCCATTTGTACTGCTG-3' (SEQ ID NO: 7 in the sequence Listing);

the second set of primer combinations comprises a first round of amplification primers, a second round of amplification primers and a sequencing primer:

the first round amplification primers comprise:

reverse primer: 5 '-CTGCTATGTYGRCACCCAATTCTG-3' (SEQ ID NO: 8 in the sequence Listing);

the second round amplification primers comprise:

reverse primer: 5 '-GAGACTCCMTGRCCCAAWTGCC-3' (SEQ ID NO: 9 in the sequence listing);

the sequencing primer comprises:

reverse primer 1: 5 '-GAGACTCCMTGRCCCAAWTGCC-3' (SEQ ID NO: 10 in the sequence Listing);

forward primer 2: 5 '-GGVATTCCCTACAATCCCCAAAG-3' (SEQ ID NO: 6 in the sequence Listing);

reverse primer 2: 5'-GAATACTGCCATTTGTACTGCTG-3' (SEQ ID NO: 7 in the sequence Listing).

In a second aspect, the invention provides a kit for detecting HIV-1 integrase gene mutation, wherein the kit comprises a first group of primer combinations and/or a second group of primer combinations in the primer group for detecting HIV-1 integrase gene mutation provided by the first aspect of the invention.

Preferably, the number of moles of primers in the first set of primer combinations is the same; the number of moles of primers in the second set of primer combinations is the same.

Preferably, the kit also contains at least one of RT-PCR amplification reagents and PCR amplification reagents.

The third aspect of the present invention provides the use of the primer set for detecting mutations in the gene of HIV-1 integrase provided according to the first aspect of the present invention or the kit for detecting mutations in the gene of HIV-1 integrase provided according to the second aspect of the present invention for preparing a preparation for detecting mutations in the gene of HIV-1 integrase.

The fourth aspect of the present invention provides a primer set for detecting mutations in the gene of HIV-1 integrase according to the first aspect of the present invention, or a kit for detecting mutations in the gene of HIV-1 integrase according to the second aspect of the present invention, or an agent for detecting mutations in the gene of HIV-1 integrase according to the third aspect of the present invention, for use in detecting or aiding in the detection of mutations in the gene of HIV-1 integrase.

In a fifth aspect, the present invention provides a method for detecting mutations in the gene of an HIV-1 integrase, the method comprising:

the method comprises the steps of sequentially carrying out reverse transcription, first round PCR amplification and second round PCR amplification by using RNA (ribonucleic acid) extracted from a sample to be detected as a template to obtain a PCR amplification product by using a primer group for detecting the mutation of the HIV-1 integrase gene provided by the first aspect of the invention, a kit for detecting the mutation of the HIV-1 integrase gene provided by the second aspect of the invention or a first group primer combination and/or a second group primer combination in a preparation for detecting the mutation of the HIV-1 integrase gene provided by the third aspect of the invention;

sequencing the PCR amplification product to obtain a sequencing result of the PCR amplification product;

and comparing the sequencing result of the PCR amplification product with an Acquired Immune Deficiency Syndrome (AIDS) virus resistance database of Stanford university to obtain HIV-1 integrase gene mutation site information.

The primer group for detecting gene mutation of the HIV-1 integrase can be used for detecting samples with the virus load of more than 1000 copies/mL, and also covers integrase-related drug-resistant sites in a Stanford drug-resistant database. The primer group for detecting the gene mutation of the HIV-1 integrase is adopted to detect the gene mutation of the HIV-1 integrase, an amplified product covers an integrase 1-279 fragment or the full length, contains all integrase drug-resistant related gene mutation sites in a Stanford HIV db database, can efficiently amplify a sample of 1000 copies per milliliter, and ensures the detection of HIV-1 epidemic strains in China, thereby providing an effective and economic solution for the localization of HIV-1 integrase drug-resistant detection reagents and the molecular epidemiological investigation, and the method has the advantages of high accuracy for detecting the gene mutation of the integrase, good amplification sensitivity, high accuracy, high repeatability, lower cost, suitability for popularization and use, and important significance for the prevention and treatment of AIDS.

Drawings

FIG. 1 shows the positions of integrase genes detected using the primer sets and methods provided by the present invention.

FIG. 2 shows the result of electrophoretic identification of the second round PCR amplification product after the first set of primer combination is used in example 3.

FIG. 3 is a diagram of the phylogenetic tree Neighbor-Joining based on the integrase sequence for the accuracy test in example 4; IN the figure, IH after sequence number "_" indicates the sequence generated using the method of the invention, VS indicates the sequence generated by Viroseq IN kit.

FIG. 4 is a diagram showing the sequence of integrase-based Neighbor-Joining phylogenetic tree in the accuracy evaluation test in example 4; in the figure, the number after the sequence number "_" is the sequence mark generated by parallel detection.

FIG. 5 is a diagram showing the phylogenetic tree Neighbor-Joining based on the integrase sequence in the reproducibility evaluation test in example 4; in the figure, the first number of the sequence number is the number of repetitive detections.

Detailed Description

In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific embodiments. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Various reagents, materials and the like used in the following examples are commercially available products unless otherwise specified; unless otherwise specified, all the tests and detection methods used in the following examples are conventional in the art and can be obtained from textbooks, tool books or academic journals.

Example 1

This example illustrates the design and optimization of primers.

Downloading an HIV-1 full-length sequence in an HIV database (database), selecting front and rear parts of an integrase region based on a B.FR.83.HXB (K03455) sequence, selecting conserved regions to design primers, analyzing the specificity of the primers by using QuickAlign software and contrasting 5 main epidemic subtypes B, CRF01_ AE, CRF07_ BC, CRF _08BC and CRF55_01B of China, analyzing a dimer and hairpin structure of the primers by using Primer 5 software, calculating the GC content and annealing temperature of the primers, carrying out experimental detection, and selecting an optimal Primer combination. The selected primer combination simultaneously considers the subtype C and part of the subtype A.

The position of the integrase gene to be detected in the present invention is located in the end of the 4230-5068 (HXB 2) pol gene region of the HIV-1 genome, as shown in FIG. 1.

The primers used include two groups, and one group may be optionally used as needed, and preferably the first group primer combination is used, and further preferably both the first group primer combination and the second group primer combination are used.

In the following primer sequences, the letter "A" represents "adenine", the letter "T" represents "thymine", the letter C represents "cytosine", and the letter G represents "guanine"; the letters R \ Y \ K \ M \ V all represent mixed bases, wherein: the letter "R" represents "A + G", the letter "Y" represents "C + T", and the letter "K" represents "G + T"; the letter "M" represents "A + C", the letter "V" represents "A + G + C", and the letter "W" represents "A + T". In the present invention, the letters used in the nucleic acid sequences are all as defined above unless otherwise specified. The term "mixed base" refers to two or three bases at that position, for example, a position is a mixed base "Y", that is, the mixed base "Y" of the sequence is at a position having both C and T, and the molar ratio is generally 1:1, and the incorporation probability of both dNTPs is generally 50% when the two dNTPs are added simultaneously during primer synthesis.

The first set of primer combinations amplified samples for all subtypes and the second set of primer combinations was not conserved for a portion of the CRF07_ BC subtype (i.e., amplification efficiency was not good enough for a portion of the CRF07_ BC subtype). The second group of primers are combined and amplified to form an integrase full-length region; compared with the fragments amplified by the second primer combination, the fragments amplified by the first primer combination have 28 bases less at the 3' end of the integrase, but the bases are not in the range of the drug-resistant mutation sites according to the Stanford drug-resistant database, and the drug-resistant detection is not influenced.

A first set of primer combinations:

the kit comprises a first round amplification primer, a second round amplification primer and a sequencing primer, wherein the reference HIV-1 genome sequence is HXB2, and the reference NCBI sequence number K03455.1 specifically comprises the following components:

first round amplification primers:

forward primer 1(INF12-1, corresponding to HXB2 genomic position 4059 → 4080 lateral forward):

5 '-GGRATYATTCARGCACAACCAG-3' (SEQ ID NO: 1 in the sequence Listing);

forward primer 2(INF12-2, corresponding to HXB2 genomic position 4053 → 4074 in the lateral forward direction):

5 '-GCATTAGGRATYATTCARGCAC-3' (SEQ ID NO: 2 in the sequence Listing);

reverse primer (INR15-1, corresponding to HXB2 genomic position 5192 ← 5214 outer reverse):

5 '-TGGGATRTGTACTTCYGARCTTA-3' (SEQ ID NO: 3 in the sequence listing).

Second round amplification primers:

forward primer (INF09, corresponding to HXB2 genomic position 4141 → 4164 forward inside):

5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer (INR18, corresponding to HXB2 genomic position 5068 ← 5088 inner reverse):

5 '-CATCCTGTCTACYTGCCACAC-3' (SEQ ID NO: 5 in the sequence listing).

Sequencing primer:

forward primer 1(INF09, corresponding to HXB2 genomic position 4141 → 4164 forward sequencing):

5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer 1(INR18, corresponding HXB2 genomic position 5068 ← 5088 reverse sequencing):

5 '-CATCCTGTCTACYTGCCACAC-3' (SEQ ID NO: 5 in the sequence Listing);

forward primer 2(KVL082, corresponding to HXB2 genomic position 4647 → 4669 forward sequencing):

5 '-GGVATTCCCTACAATCCCCAAAG-3' (SEQ ID NO: 6 in the sequence Listing);

reverse primer 2(KVL083, corresponding HXB2 genome position 4750 ← 4772 reverse sequencing):

5'-GAATACTGCCATTTGTACTGCTG-3' (SEQ ID NO: 7 in the sequence Listing).

A second set of primer combinations:

similar to the first set of primer combinations, the first round amplification primer, the second round amplification primer and the sequencing primer are also included, specifically as follows:

first round amplification primers:

5 '-GGRATYATTCARGCACAACCAG-3' (SEQ ID NO: 1 in the sequence Listing);

5 '-GCATTAGGRATYATTCARGCAC-3' (SEQ ID NO: 2 in the sequence Listing);

reverse primer (INR08, corresponding to HXB2 genomic position 5775 ← 5798 outer reverse):

5 '-CTGCTATGTYGRCACCCAATTCTG-3' (SEQ ID NO: 8 in the sequence Listing).

Second round amplification primers:

5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer (INR06, corresponding to HXB2 genomic position 5276 ← 5297 inside reverse):

5 '-GAGACTCCMTGRCCCAAWTGCC-3' (SEQ ID NO: 9 in the sequence listing).

Sequencing primer:

5 '-TCTAYCTGKCATGGGTRCCAGCAC-3' (SEQ ID NO: 4 in the sequence listing);

reverse primer 1(INR06, corresponding to HXB2 genomic position 5276 ← 5297 reverse sequencing):

5 '-GAGACTCCMTGRCCCAAWTGCC-3' (SEQ ID NO: 10 in the sequence Listing);

forward primer 2(KVL082, forward sequencing of 4647-4669 at HXB2 genomic position):

5 '-GGVATTCCCTACAATCCCCAAAG-3' (SEQ ID NO: 6 in the sequence Listing);

reverse primer 2(KVL083, reverse sequencing of 4750-4772 corresponding to HXB2 genome position):

5'-GAATACTGCCATTTGTACTGCTG-3' (SEQ ID NO: 7 in the sequence Listing).

Example 2

This example illustrates the extraction of RNA samples.

The source of the material used to extract RNA in this example is serum or plasma from the patient to be tested for HIV-1 (hereinafter collectively referred to as "sample material").

Before RNA extraction, the sample material was removed from the freezer at-80 ℃ in advance, returned to room temperature (15-25 ℃) and thawed for use.

RNA extraction is carried out by using a QIAGEN Viral RNA Mini Kit virus RNA extraction Kit, and before operation, the AVE buffer solution in the Kit is returned to room temperature for subsequent steps. The specific operation steps for extracting RNA are as follows:

1. adding 1.5mL of AVL buffer solution into a tube of freeze-dried Carrier RNA, transferring the solution into an AVL buffer solution bottle (one tube of Carrier RNA corresponds to 31mL of AVL) after the solution is completely dissolved, and subpackaging the solution into 1.5mL eppendorf tubes with 560 μ L per tube for storage at 4 ℃. The AVL/Carrier RNA buffer solution has precipitation when stored at 4 ℃, and the precipitation needs to be dissolved by preheating at 37 ℃ before use. Note: the AVL/CarrierRNA buffer was not heated more than 6 times and each incubation was not allowed to proceed for more than 5 minutes.

AW1 and AW2 buffers were added to 96-100% ethanol, in the amounts specified, the ethanol being free of RNase (absolute ethanol).

All centrifugation steps were performed at room temperature. Note that: tip heads without DNase and RNase were used.

2. 560. mu.L of prepared AVL buffer containing Carrier RNA was added to a 1.5mL centrifuge tube.

3. Add 140. mu.L of plasma to the tube containing AVL/Carrier RNA buffer and mix by shaking for 15 seconds.

4. Incubate at room temperature (15-25 ℃) for 10 minutes.

5. A1.5 mL centrifuge tube was briefly centrifuged to remove the droplets in the tube cap.

6. 560. mu.L of ethanol (96-100%) was added and mixed by shaking for 15 seconds. After oscillation and uniform mixing, the centrifugal tube is centrifuged for a short time to eliminate liquid drops in the tube cover.

7. 630 μ L of the solution from step 6 was carefully transferred to a QIAamp RNA extraction column (placed in a 2mL collection tube) taking care not to touch the edge of the column wet. The lid was closed, centrifuged at 6000g (approx. 8000 rotations in a small centrifuge) for 1 minute, the column was transferred to a new 2mL collection tube (provided), and the collection tube with the remaining filtrate was discarded.

8. Carefully open the lid of the column and repeat step 7.

9. Carefully open the lid of the column, add 500. mu.L AW1 solution, cover the lid, centrifuge at 6000g (8000rpm) for 1 minute, place the column in a new 2mL collection tube, and discard the collection tube of the remaining filtrate.

10. Carefully open the column cover, add 500. mu.L AW2 solution, cover the lid, and centrifuge at 20000g full speed (14000rmp) for 3 minutes.

11. The column was placed into a new 1.5mL centrifuge tube (self-contained) and the collection tube with the remaining filtrate was discarded. Carefully open the column cover, add 60. mu.L of AVE buffer that has returned to room temperature, cover, incubate at room temperature for 5 minutes, 6000g (8000rmp) centrifugal 1 minutes.

12. And centrifuging to obtain an eluent, namely the RNA sample. Note that: the RNA sample is easy to degrade, subsequent RT-PCR reaction is required to be carried out immediately, if the RT-PCR cannot be carried out immediately, the RNA sample can be stored at the temperature of minus 20 ℃ (one month), and can be stored at the temperature of minus 80 ℃ (half a year) for a long time.

Example 3

This example illustrates RT-PCR amplification, sequencing and analysis.

1. Reverse transcription and first round PCR

The method adopts Promega company with A1702 Access quick^TMRT-PCR System kit.

The reaction system (total volume 25. mu.L) was as follows:

wherein, the PCR Mix contains Tfl DNA polymerase, dNTPs, magnesium sulfate and reaction buffer. The forward primer 1, the forward primer 2 and the reverse primer are any one of the first round amplification primers in the two primer combinations listed in the embodiment 1 (namely, the forward primer 1 is a sequence 1 in a sequence table, the forward primer 2 is a sequence 2 in the sequence table and the reverse primer is a sequence 3 in the sequence table, or the forward primer 1 is a sequence 10 in the sequence table, the forward primer 2 is a sequence 11 in the sequence table and the reverse primer is a sequence 12 in the sequence table); the final concentration of each primer was 20. mu. mol/L.

Preparing the PCR reaction system, performing ice operation, adding template RNA (the template RNA is the RNA sample extracted according to the method in the embodiment 2), uniformly mixing by oscillation, performing short-time centrifugation, and then performing amplification according to the following PCR reaction program:

45 minutes at 50 ℃;

2 minutes at 95 ℃;

3 cycles of 95 ℃ for 20 seconds, 50 ℃ for 30 seconds, and 72 ℃ for 2 minutes and 30 seconds;

35 cycles of 95 ℃ for 15 seconds, 50 ℃ for 20 seconds, and 72 ℃ for 2 minutes;

10 minutes at 72 ℃;

storing at 4 ℃.

2. Second round PCR

This step was carried out using a kit having the product number KT201 from TIANGEN.

The reaction system (total volume 50. mu.L) was as follows:

wherein the PCR Mix premixed reagent contains Taq DNA polymerase, dNTPs and MgCl with 2 times of final concentration₂Reaction buffer solution, enhancer and optimizer of PCR reaction and stabilizer. The template is the reaction product obtained in the first round of PCR reaction. The forward primer and the reverse primer are selected from one of the two primer combinations listed in example 1 and correspond to the first round amplification primer (namely, when the first round PCR uses the sequence 1, the sequence 2 and the sequence 3 in the sequence table, the primers used in the second round PCR are the sequence 4 and the sequence 5 in the sequence table, and when the first round PCR uses the sequence 10, the sequence 11 and the sequence 12 in the sequence table, the primers used in the second round PCR are the sequence 13 and the sequence 14 in the sequence table); the final concentration of the primers was 20. mu. mol/L.

Preparing the PCR reaction system, performing ice operation, adding a template, oscillating, uniformly mixing, centrifuging for a short time, and then performing amplification according to the following PCR reaction procedures:

4 minutes at 95 ℃;

3 cycles of 95 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 2 minutes and 30 seconds;

35 cycles of 95 ℃ for 15 seconds, 55 ℃ for 20 seconds, and 72 ℃ for 2 minutes;

10 minutes at 72 ℃;

storing at 4 ℃.

3. Electrophoretic identification of second round PCR amplification product

(1) Weighing 1.5g of agarose in a triangular flask, adding 150mL of 1 XTAE solution to prepare 1% gelatin solution, placing the gelatin solution in a microwave oven, heating for 3 minutes by using a medium fire, adding 5 mu L of Gold View dye when the gelatin solution is cooled to about 60 ℃, and uniformly mixing.

(2) The cleaned electrophoresis plate and the comb are wiped dry, and the sol solution is poured into the electrophoresis plate to ensure that the thickness of the gel reaches about 0.5 cm. Standing at room temperature for at least 40 min to solidify the gel completely.

(3) And slowly pulling out the comb after the gel is finished, putting the comb into an electrophoresis tank, and pouring 1 × TAE into the electrophoresis tank to ensure that the liquid level is about 1mm higher than the plane of the gel.

(4) And taking 5 mu L of the second round PCR product and slowly adding the product into the electrophoresis hole. (if the reaction system does not contain a Loading Buffer, dropping 10 multiplied by sample Buffer solution on the glass paper, each drop being about 1 mu L, taking 5 mu L of the second round PCR amplification product and 10 multiplied by sample Buffer solution drops, mixing uniformly, and slowly adding into the electrophoresis hole.) and adding 5 mu L of Marker with molecular weight of 2000 into the electrophoresis hole, and judging the correct position of the PCR amplification product.

(5) The voltage setting of the electrophoresis apparatus is 100V, the electrophoresis is carried out at constant voltage, and the result is observed in about 40 minutes.

(6) And taking the gel after electrophoresis off the gel plate, placing the gel plate on a gel imager, observing bands of expected PCR products in the gel, and taking pictures.

The results of using the first set of primer combinations are shown in fig. 2. In fig. 2, GEN201911.01A, GEN201911.02A, GEN201911.03A, GEN201911.04A, GEN201911.05A are standard samples from the drug resistance quality control program of the us VQA (virology quality assurance) laboratory; ZK6, ZK31, and ZK54 were clinical samples, and are specifically shown in Table 4.

(7) And cutting a gel strip of the positive sample around 948bp, purifying and recovering a DNA fragment for subsequent sequencing. At the same time, the first round PCR product was stored at-20 ℃ for re-amplification if necessary.

4. Sequencing and sequence analysis

Sequencing the DNA fragment obtained by the recovery by a Sanger sequencing method.

The primers used for sequencing are sequencing primers corresponding to the first round amplification primers and the second round amplification primers in the two primer combinations listed in example 1 (namely, when the first round PCR uses the sequence 1, the sequence 2 and the sequence 3 in the sequence table, and the second round PCR uses the sequence 4 and the sequence 5 in the sequence table, the sequencing primers use the sequence 6, the sequence 7, the sequence 8 and the sequence 9 in the sequence table, when the first round PCR uses the sequence 10, the sequence 11 and the sequence 12 in the sequence table, and when the second round PCR uses the sequence 13 and the sequence 14 in the sequence table, the sequencing primers use the sequence 15, the sequence 16, the sequence 17 and the sequence 18 in the sequence table).

5. Sequence analysis

HIV-1 integrase gene mutation site information is obtained by comparing the HIV-integrase gene mutation site information with an acquired immune deficiency syndrome virus resistance database of Stanford university.

Example 4

This example illustrates the accuracy, amplification sensitivity, accuracy and reproducibility of the detection method for detecting mutations in integrase gene of HIV-1 virus provided by the present invention.

In this embodiment, the primers used are two groups as in embodiment 1, the first group of primer combination is used for detection, and then the second group of primers is used for detection, and the final detection result is obtained when the two groups of primers are adopted, so that the reliability of the detection result is improved.

(I) detection accuracy

The accuracy is measured by comparing the nucleotide sequence generated by the detection method of the invention with the nucleotide sequence generated by the gold standard method and the drug resistance mutation result. Using ViroSeq^TMHIV-1 integrase gene drug-resistant detection reagentBox (ViroSeq)^TMIN kit) is a gold standard method, which is manufactured by yapei corporation, usa, and although the IN kit is used only for Research Use (RUO), and is not approved by FDA or certified by CE-IVD, it is a more recognized commercial kit IN the market at present, and thus is often used as a "gold standard" IN the method alignment.

The pass standard: sequence identity obtained by both methods was 98% and above for 90% and above samples.

Description of the drawings: inconsistencies (i.e., differences) include: partial inconsistency (compatibility) and complete inconsistency (incompatibility); when compatibility is different, one sequence is a mixed base, and the corresponding base in the other sequence is one base in the mixed base, such as one is R, and the other is A; when the incompatibility is different, the corresponding bases of the two sequences are completely different, such as one is A, and the other is T.

1. Sample source

A total of 26 samples were included in the comparison for accuracy evaluation, including:

(1) clinical samples: samples from 11 plasma samples from chinese patients were used as clinical samples with a viral load between 6000 and 650000 copies/ml.

(2) Analyzing a sample: samples of 15 VQA virus quality control programs from NIH in the united states were referred to as assay samples, with viral loads between 5766 and 26833 copies/ml.

These samples covered 9 subtypes: a1(1), B (10), C (2), CRF01_ AE (5), CRF02_ AG (1), CRF07_ BC (1), CRF08_ BC (4), CRF55_01B (1), F1 (1). Sample conditions are shown in table 1.

TABLE 1 sample case for accuracy evaluation

2. Sequence identity comparison

The detection method and the use of Viroseq^TMThe sequences generated by the IN kit were found by Neighbor-join phylogenetic tree analysis, and the sequences generated by the same sampleRows are clustered together, gene distance<0.015, the results are shown in FIG. 3.

The sequences are pairwise matched and compared, and the result shows that the sequence consistency of all samples is more than 98.6 percent, wherein the number of inconsistent bases is between 0 and 11, and only one sample has 1 completely inconsistent base (Table 2), which indicates that the method provided by the invention can obtain high detection accuracy.

TABLE 2 detection methods and Viroseq of the invention^TMPairing comparison of IN kit parallel detection sequences

(II) sensitivity of amplification

The amplification sensitivity is the PCR amplification success rate, namely the amplification success rate in samples covering main popular subtypes in China in a certain virus load range, the samples comprise clinical (undiluted) samples and analytical (diluted) samples, and the number of the samples with the load of 1000-5000 copies/ml is more than 10.

By standard: the sample with 90% loading of 1000-5000 copies/ml was successfully amplified, and the sample with 95% loading of >5000 copies/ml was successfully amplified.

1. Sample composition

43 clinical specimens: the viral load ranged from 1098-543115 copies/ml, encompassing six HIV-1 subtypes, including B' (3), CRF01_ AE (16), CRF07_ BC (17), CRF08_ BC (1), CRF55_01B (1), CRF59_01B (1), and URF (4).

3 clinical samples: viral loads ranged between 58-3830 copies/ml, subtypes B', CRF01_ AE and CRF07_ BC, respectively.

14 samples analyzed: 14 samples were analyzed by dilution with HIV-negative plasma to a viral load of 1000-5000 copies per ml.

2. The result of amplification

8 samples (72%, 8/11) with the virus load of 51-1000 copies/mL are successfully amplified, 16 samples (94%, 16/17) with the virus load of 1001-5000 copies/mL are successfully amplified, and all samples with the virus load of more than 5000 copies/mL are successfully amplified (Table 3), which shows that the method provided by the invention can obtain high amplification sensitivity.

TABLE 3 viral load Range and amplification of samples for amplification sensitivity evaluation

(III) evaluation of accuracy

Accuracy was assessed by comparison of the results of the in-batch tests. Selecting 4 clinical samples, extracting virus RNA from each sample to sequence in the same batch for 5 parallel detections, constructing a Neighbor-join evolutionary tree by using Mega X, performing pairwise comparison by using WHO HIVDR QC online software (https:// call.

By standard: the 90% consistency of the pairwise aligned sequences is more than 98%, and partial inconsistency is counted as inconsistency.

1. Sample condition

The viral load of 4 clinical specimens was between 8800 and 28100, and the subtypes included: b' (1), CRF01_ AE (2) and CRF07_ BC (1), all without resistance mutations, see table 4.

TABLE 4 basic and Overall evaluation of samples for accuracy evaluation

2. The evolutionary tree analysis shows that the sequences of the same sample are all located on the same branch, and the genes are close to each other, and the result is shown in FIG. 4.

3. Consistency comparison

The total number of base differences between the sequences of a single sample was between 0 and 40, with an average identity between 99.52% and 100% (Table 5). Pairwise comparison shows that the differences between the two sequences are between 0 and 7 bases, which are caused by mixed bases and have no completely inconsistent bases (table 5), which indicates that very high consistency can be obtained by using the method provided by the invention.

TABLE 5 parallel determination of the case of pairwise comparison of sample sequences

(IV) evaluation of reproducibility

Repeatability is assessed by comparison of the results of the test between batches. 4 clinical specimens were selected, and two operators performed the test at least once every month using different batches of viral RNA extraction reagents (both viral RNA extraction kits purchased from QIAGEN), and 5 batches of test were performed per specimen (Table 6).

As described above, the Neighbor-join evolutionary tree was constructed using Mega X, and the nucleotide sequence similarity was calculated using WHO HIVDR QC online software (https:// call.

TABLE 6 reagents, assay times and operator profiles for reproducible evaluation

1. Sample conditions were consistent with the samples used for accuracy evaluation (table 7).

TABLE 7 basic and overall evaluation of samples for repeatability evaluation

2. The evolutionary tree analysis shows that the sequences of the same sample are all located on the same branch, and the genes are close to each other, and the result is shown in FIG. 5.

3. Consistency comparison

A total of 10 pairwise comparisons were performed for each sample, with a total number of base differences between 6 and 40 and an average difference between 0.1 and 0.9% (Table 7). Pairwise comparison shows that the differences between the two sequences are between 0 and 10 bases, and are caused by mixed bases, and no completely unmatched differences exist (Table 8), which indicates that the method provided by the invention has high repeatability.

TABLE 8 repeated detection of the case of pairwise comparison of sample sequences

In conclusion, the method for detecting the gene mutation of the integrase of the HIV-1 virus meets the standards recommended by WHO in the aspects of accuracy, amplification sensitivity, accuracy and repeatability, and can be completely suitable for the drug resistance detection of HIV-1 virus strains popular in China to the integrase.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> prevention and control center of Chinese diseases for preventing and controlling venereal disease and AIDS

Beijing Anpu Biochemical Technology Co.,Ltd.

<120> primer set for detecting HIV-1 integrase gene mutation and application thereof

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Claims

1. A primer set for detecting gene mutation of an HIV-1 integrase, wherein the primer set comprises a first set of primer combination and a second set of primer combination;

the first round amplification primers comprise:

forward primer 1: 5 '-GGRATYATTCARGCACAACCAG-3', namely the sequence 1 in the sequence table;

forward primer 2: 5 '-GCATTAGGRATYATTCARGCAC-3', namely the sequence 2 in the sequence table;

reverse primer: 5 '-TGGGATRTGTACTTCYGARCTTA-3', namely the sequence 3 in the sequence table;

the second round amplification primers comprise:

a forward primer: 5 '-TCTAYCTGKCATGGGTRCCAGCAC-3', namely the sequence 4 in the sequence table;

reverse primer: 5 '-CATCCTGTCTACYTGCCACAC-3', namely the sequence 5 in the sequence table;

the sequencing primer comprises:

forward primer 1: 5 '-TCTAYCTGKCATGGGTRCCAGCAC-3', namely the sequence 4 in the sequence table;

reverse primer 1: 5 '-CATCCTGTCTACYTGCCACAC-3', namely the sequence 5 in the sequence table;

forward primer 2: 5 '-GGVATTCCCTACAATCCCCAAAG-3', namely the sequence 6 in the sequence table;

reverse primer 2: 5'-GAATACTGCCATTTGTACTGCTG-3', sequence 7 in the sequence table;

the first round amplification primers comprise:

forward primer 1: 5 '-GGRATYATTCARGCACAACCAG-3', namely sequence 1 in the sequence table;

forward primer 2: 5 '-GCATTAGGRATYATTCARGCAC-3', namely sequence 2 in the sequence table;

reverse primer: 5 '-CTGCTATGTYGRCACCCAATTCTG-3', namely the sequence 8 in the sequence table;

the second round amplification primers comprise:

reverse primer: 5 '-GAGACTCCMTGRCCCAAWTGCC-3', namely the sequence 9 in the sequence table;

the sequencing primer comprises:

reverse primer 1: 5 '-GAGACTCCMTGRCCCAAWTGCC-3', namely the sequence 10 in the sequence table;

reverse primer 2: 5'-GAATACTGCCATTTGTACTGCTG-3', i.e., SEQ ID NO. 7 of the sequence Listing.

2. A kit for detecting HIV-1 integrase gene mutation, comprising the first set of primer combination and/or the second set of primer combination in the primer set for detecting HIV-1 integrase gene mutation according to claim 1.

3. The kit of claim 2, wherein the number of moles of primers in the first set of primer combinations is the same; the number of moles of primers in the second set of primer combinations is the same.

4. The kit of claim 2, wherein the kit further comprises at least one of RT-PCR amplification reagents and PCR amplification reagents.

5. Use of the primer set for detecting mutation of HIV-1 integrase gene according to claim 1 or the kit for detecting mutation of HIV-1 integrase gene according to any one of claims 2 to 4 for preparing a preparation for detecting mutation of HIV-1 integrase gene.

6. Use of the primer set for detecting mutation of HIV-1 integrase gene according to claim 1, or the kit for detecting mutation of HIV-1 integrase gene according to any one of claims 2 to 4, or the preparation for detecting mutation of HIV-1 integrase gene according to claim 5 for detecting or assisting in detecting mutation of HIV-1 integrase gene.

7. A method of detecting mutations in the HIV-1 integrase gene, the method comprising:

using the primer set for detecting gene mutation of HIV-1 integrase according to claim 1, or the kit for detecting gene mutation of HIV-1 integrase according to any one of claims 2 to 4, or the first set of primer combination and/or the second set of primer combination in the preparation for detecting gene mutation of HIV-1 integrase according to claim 5, and sequentially performing reverse transcription, the first round of PCR amplification and the second round of PCR amplification by using RNA extracted from a sample to be detected as a template to obtain a PCR amplification product;

and comparing the sequencing result of the PCR amplification product with an acquired immune deficiency syndrome virus (AIDS) drug-resistant database of Stanford university to obtain HIV-1 integrase gene mutation site information.