CN108251500A - Detect primer and its application of the main resistant mutational site for the treatment of AIDS drug non-nucleoside reverse transcriptase inhibitor - Google Patents

Abstract

The present invention relates to for detecting the primer of the main resistant mutational site for the treatment of AIDS drug non-nucleoside reverse transcriptase inhibitor,Mutational site K103N including detecting 1 viral pol genes of HIV,V108I,E138A,Y181C,Y188C,G190A,The ASPCR primers of G190R and M230I,The sense primer in each mutational site is respectively SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3,SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.7,Nucleotide sequence shown in SEQ ID NO.8,Downstream primer is the nucleotide sequence shown in SEQ ID NO.9.The present invention is used to carry out specific amplification to treating AIDS drug NNRTIs resistant mutational sites, has the advantages that high specificity, high sensitivity, simplicity are quick, at low cost.

Description

Detection of major drug resistance mutations of non-nucleoside reverse transcriptase inhibitors for HIV treatment Primers for loci and their applications

technical field

The invention relates to the technical fields of molecular biology and biomedicine, in particular to a primer for detecting main drug-resistant mutations of non-nucleoside reverse transcriptase inhibitors, an AIDS treatment drug.

Background technique

AIDS is an infectious disease caused by the human immunodeficiency virus (HIV). The HIV virus destroys the immune system of the infected person, and eventually various opportunistic infections and tumors appear. According to the difference of HIV gene, it can be divided into two types: HIV-1 and HIV-2, and HIV-1 infection is the main type.

At present, there is no cure for AIDS. The most commonly used is highly active combined antiretroviral therapy (HAART), which can effectively inhibit virus replication and control the virus content in the infected person to a level that cannot be detected by existing methods. There are at least 34 antiviral drugs in 6 categories, including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors (FIs), Viral entry inhibitors (EIs), integrase inhibitors (INs).

There are 9 types of antiviral drugs provided by the Chinese government free of charge, including 5 NRTIs, 2 NNRTIs, and 2 PIs. The first-line antiviral treatment generally consists of 2 NRTIs and 1 NNRTI. During the course of treatment, the following reasons combined to lead to the emergence of drug resistance: (1) HIV-1 rapid replication and lack of proofreading function of reverse transcriptase, poor fidelity during the replication process, and a large number of mutations in gene sequences; (2) drug efficacy (3) The genetic factors and compliance of the host. Under the pressure of drug selection, the strains containing drug-resistant mutations gradually become dominant strains, and their sensitivity to therapeutic drugs decreases, resulting in incomplete viral suppression or failure of antiviral therapy.

At present, HIV drug resistance detection methods are mainly divided into genotype detection and phenotype detection. Phenotype detection is the gold standard for HIV-1 drug resistance detection. To determine the impact of HIV-1 replication in the presence of drugs, patients need to obtain The virus was isolated and infected peripheral blood mononuclear cells, and the half inhibitory concentration (IC ₅₀ ) of the drug was determined to evaluate the drug resistance. The experimental process requires high technology, long cycle, and high price. It is generally used to evaluate the virus's sensitivity to new drugs or the impact of drug-resistant sites on drug sensitivity.

Genotype detection is to obtain the gene sequence of the virus by sequencing, and analyze whether there are drug resistance-related mutations in the sequence to determine the drug resistance of the virus strain. The method involves steps such as viral RNA extraction, reverse transcription, PCR amplification, sequencing, and sequence analysis. It has the characteristics of simple operation and low cost, and is currently a commonly used method in drug resistance monitoring. However, genotype detection also has certain limitations: (1) The laboratory cannot independently complete the entire experimental process, and the PCR-amplified products need to be sent to a sequencing company for sequencing, which makes the experimental cycle long and has certain limitations on clinical applications; (2) Traditional Sanger sequencing obtains the common sequence of quasispecies, and cannot detect the inferior strains with a content of less than 20%, and loses the sequence information of some low-abundance drug-resistant strains. Therefore, it is necessary to establish a drug resistance detection method with simple operation, short analysis cycle, accurate results, and low cost for large-scale drug resistance monitoring.

Allele-specific PCR (ASPCR) is a method for SNP typing. A SNP site includes two specific primers and a universal primer. The 3' ends of the two specific primers are respectively aligned with the SNP The base pairing of the two alleles, the specific primer can amplify the genotype that matches it but cannot amplify the genotype that does not match it, so the SNP type can be judged by the result of PCR amplification.

However, no one has yet designed ASPCR primers to detect major resistance mutations to HIV treatment drugs such as non-nucleoside reverse transcriptase inhibitors.

Contents of the invention

The technical problem to be solved by the present invention is to provide a primer for rapid and sensitive detection of main drug-resistant mutation sites of non-nucleoside reverse transcriptase inhibitors of AIDS treatment drugs by using PCR method and its application.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a primer for detecting the main drug-resistant mutation site of non-nucleoside reverse transcriptase inhibitors for the treatment of AIDS, including the detection of the mutation site of HIV-1 virus pol gene Allele-specific PCR primers for K103N, V108I, E138A, Y181C, Y188C, G190A, G190R and M230I, each mutation site includes a specific upstream primer and a universal downstream primer, wherein,

The upstream primer and downstream primer of the K103N site are the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the V108I site are the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the E138A site are the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the Y181C site are the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the Y188C site are the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the G190A site are the nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the G190R site are the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.9 respectively;

The upstream primer and downstream primer of the M230I site are the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

The HIV-1 virus pol gene is the nucleotide sequence shown in SEQ ID NO.10.

It should be noted that the primers of the present invention are aimed at the mutation sites K103N, V108I, E138A, Y181C, Y188C, G190A, G190R, M230I of the HIV-1 virus pol gene, and can be used for K103N, V108I, E138A, Y181C, Y188C, G190A , G190R, M230I mutation sites for specific amplification, so it can be understood that these primers are not only used for the real-time PCR detection of the present invention, but also suitable for other detections based on PCR amplification, such as conventional PCR, gene chip Wait.

The invention also discloses a PCR detection method for detecting mutation sites K103N, V108I, E138A, Y181C, Y188C, G190A, G190R, M230I of the HIV-1 virus pol gene, the detection method comprises the primers designed in the present invention, with The cDNA of the sample to be detected is used as a template for PCR amplification. The reaction system for real-time fluorescent PCR detection is 20uL in total, including 10uL of 2XSYBR Green PCR Master Mix, 0.4uL of upstream and downstream primers, 2uL of cDNA template for the sample to be tested, and 7.2uL of ddH ₂ O. The reaction program is: ①pre-denaturation at 95°C for 1 min, ②then at 95°C for 10 sec, annealing at 62°C for 30 sec, and extension at 72°C for 1 min, repeating 40 cycles, ③at a rate of 0.5°C every 5 seconds, from 60°C to 95°C °C, the specific dissolution peak of the product was obtained.

The real-time fluorescent PCR detection of the main drug-resistant mutations of the AIDS treatment drug NNRTIs includes the following three aspects of detection content:

(1) Specificity test: Real-time real-time PCR detection was carried out using the HIV-1 pol gene wild-type plasmid and mutant plasmid as templates and using specific primers for the corresponding sites.

(2) Sensitivity test: Dilute the mutant plasmid template to 1ng/uL, 10 ^-1 ng/uL, 10 ^-2 ng/uL, 10 ^-3 ng/uL, 10 ^-4 ng/uL, 10 ^-5 ng/uL uL, using the specific primers corresponding to the site, for real-time real-time PCR detection.

The method for detecting main drug-resistant mutations of AIDS treatment drugs NNRTIs based on the allele-specific PCR method disclosed by the invention has the advantages of strong specificity, high sensitivity, simplicity, speed, and low cost. The designed ASPCR primers are only aimed at the base mutation sequence at a specific site, can specifically amplify the mutation template, and the detection sensitivity of the gene mutation can reach 10% (that is, the number of mutant gene RNA templates of the target gene accounts for 10% of the number of wild-type RNA templates) , while the sensitivity of the direct sequencing method to detect gene mutations is 10% (that is, the number of mutated gene RNA templates of the target gene accounts for 10% of the number of wild-type RNA templates). The invention can effectively detect the low-abundance drug resistance of AIDS, and overcomes the disadvantages of long experiment cycle and high detection abundance of the sequencing method. The invention only involves the common method of real-time detection, which is easy to grasp and fast to operate, and can judge the gene type according to the CT value of PCR amplification.

Description of drawings

Fig. 1 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the K103N site mutant plasmid as templates and K103N site ASPCR primers respectively.

Fig. 2 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the V108I site mutant plasmid as templates, and the V108I site ASPCR primers respectively.

Fig. 3 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the E138A site mutant plasmid as templates, and E138A site ASPCR primers respectively.

Fig. 4 is a diagram of specificity test PCR results of HIV-1 wild-type cloning plasmid and Y181C site mutant plasmid as templates and Y181C site ASPCR primers respectively.

Fig. 5 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the Y188C site mutant plasmid as templates, and the Y188C site ASPCR primers respectively.

Fig. 6 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the G190A site mutant plasmid as templates, and the G190A site ASPCR primers respectively.

Fig. 7 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the G190R site mutant plasmid as templates, and the G190R site ASPCR primers respectively.

Fig. 8 is a graph showing the specificity test PCR results of the HIV-1 wild-type cloning plasmid and the M230I site mutant plasmid as templates, and the M230I site ASPCR primers respectively.

Fig. 9 is a graph showing the sensitivity test PCR results of K103N site ASPCR primers with different ratios of K103N mutation samples as templates.

Fig. 10 is a graph showing the PCR results of the sensitivity test of ASPCR primers at the V108I site with different ratios of V108I mutation samples as templates.

Fig. 11 is a graph showing the PCR results of the sensitivity test of ASPCR primers at the E138A site using different ratios of E138A mutation samples as templates.

Fig. 12 is a graph showing the sensitivity test PCR results of ASPCR primers at the Y181C site with different ratios of Y181C mutation samples as templates.

Fig. 13 is a graph showing the PCR results of the sensitivity test of the ASPCR primers at the Y188C site using different ratios of Y188C mutation samples as templates.

Fig. 14 is a graph showing the sensitivity test PCR results of ASPCR primers at the G190A site using different proportions of G190A mutation samples as templates.

Fig. 15 is a graph showing the sensitivity test PCR results of ASPCR primers at the G190R site using different proportions of G190R mutation samples as templates.

Fig. 16 is a graph showing the sensitivity test PCR results of ASPCR primers at the M230I site using different ratios of M230I mutation samples as templates.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are used to illustrate the present invention and not to limit the scope of the present invention.

Embodiment 1: Construction of HIV-1 wild-type plasmid and mutant plasmid

(1) Ligate the reverse transcriptase gene sequence (length 1.1kb, relative to HXB2 position: 2243-3304) identified by sequencing to contain no drug-resistant mutations with the pUCm-19 vector, then transform, extract plasmid DNA, and verify plasmid connection by sequencing Correct, HIV-1 wild-type cloning plasmids were obtained.

(2) By artificial site-directed mutagenesis, mutate the bases corresponding to amino acids 103, 108, 138, 181, 188, 190, and 230 of the reverse transcription gene in the HIV-1 wild-type cloning plasmid to obtain K103N and V108I , E138A, Y181C, Y188C, G190A, G190R, M230I site mutant plasmids.

Embodiment 2: Primer specificity test

(1) Using the HIV-1 wild-type cloning plasmid and the K103N site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.1 and SEQ ID NO.9. The reaction system for real-time fluorescent PCR detection is 20uL in total, including 10uL of 2XSYBR Green PCR Master Mix, 0.4uL of 10 ⁴ nmol/L upstream and downstream primers, 2uL of 10 ⁴ copies/uL template, and 7.2uL of ddH ₂ O. The reaction program was: ①pre-denaturation at 95°C for 1min, ②then at 95°C for 10sec, annealing at 62°C for 30sec, and extension at 72°C for 1min, repeating 40 cycles. The result is shown in Figure 1. The present invention can directly interpret the results of the amplification curve of real-time fluorescent PCR. The amplification curve (marker 1) of the K103N mutant template shows an S-shaped growth, and the CT value of the number of cycles experienced by the fluorescent signal reaching the set threshold is 24.1, which is higher than that of the wild Type template (label 2) has no amplification curve, its fluorescence signal has not reached the set threshold, and has no CT value. The difference between the two is obvious, and the amplification specificity is high.

(2) Using the HIV-1 wild-type cloning plasmid and the V108I site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.2 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 2, the amplification curve (marker 3) of the V108I mutant template showed an S-shaped growth, and the CT value was 25.9, and the wild-type template (marker 4) had no amplification curve, no CT value, and the amplification specificity was relatively low. high.

(3) Using the HIV-1 wild-type cloning plasmid and the E138A site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.3 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 3, the amplification curve (marker 5) of the E138A mutant template showed an S-shaped growth, and the CT value was 23.7, and the wild-type template (marker 6) had no amplification curve, no CT value, and the amplification specificity was relatively low. high.

(4) Using the HIV-1 wild-type cloning plasmid and the Y181C site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.4 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 4, the amplification curve (marker 7) of the Y181C mutant template showed an S-shaped growth, and the CT value was 21.4, and the wild-type template (marker 8) had no amplification curve, no CT value, and the amplification specificity was relatively low. high.

(5) Using the HIV-1 wild-type cloning plasmid and the Y188C site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.5 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 5, the amplification curve (marker 9) of the Y188C mutant template showed an S-shaped growth, and the CT value was 25.4, and the wild-type template (marker 10) had no amplification curve, no CT value, and the amplification specificity was relatively high. high.

(6) Using the HIV-1 wild-type cloning plasmid and the G190A site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.6 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 6, the amplification curve (marker 11) of the G190A mutant template showed an S-shaped growth, and the CT value was 22.5, and the wild-type template (marker 12) had no amplification curve, no CT value, and the amplification specificity was relatively high. high.

(7) Using the HIV-1 wild-type cloning plasmid and the G190R site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.7 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 7, the amplification curve (marker 13) of the G190R mutant template showed an S-shaped growth, and the CT value was 22.5, and the wild-type template (marker 14) had no amplification curve, no CT value, and the amplification specificity was relatively high. high.

(8) Using the HIV-1 wild-type cloning plasmid and the M230I site mutant plasmid as templates, real-time fluorescent PCR detection was performed using primers SEQ ID NO.8 and SEQ ID NO.9. The reaction system and reaction procedure of real-time fluorescent PCR detection are the same as (1). The results are shown in Figure 8, the amplification curve (label 15) of the M230I mutant template showed an S-shaped growth, and the CT value was 25.0, and the wild-type template (label 16) had no amplification curve, no CT value, and amplification specificity higher.

Embodiment 3: Primer Sensitivity Test

(1) Take 10 ⁴ copies/uL HIV-1 wild-type cloning plasmid and 10 ⁴ copies/uL K103N site mutant plasmid and mix them in different ratios to prepare K103N mutant samples with different ratios to obtain 1% mutant samples (mutant plasmid and The ratio of wild type plasmid is 1:100), 5% mutant samples (ratio of mutant plasmid to wild type plasmid is 5:100), 10% mutant samples (ratio of mutant plasmid to wild type plasmid is 10:100) , 100% mutant samples (without wild-type plasmid). The primers SEQ ID NO.1 and SEQ ID NO.9 were used as primers, and the above-mentioned 1% mutant samples, 5% mutant samples, 10% mutant samples, and 100% mutant samples were used as templates to carry out real-time fluorescent PCR reaction. The reaction system for real-time fluorescent PCR detection is 20uL in total, including 10uL of 2X SYBR Green PCR Master Mix, 0.4uL of 10 ⁴ nmol/L upstream and downstream primers, 2uL of template, and 7.2uL of ddH ₂ O. The reaction program was: ①pre-denaturation at 95°C for 1min, ②then at 95°C for 10sec, annealing at 62°C for 30sec, and extension at 72°C for 1min, repeating 40 cycles. The results are shown in Figure 9, the amplification curve of the 10% mutation sample is a typical S-shaped growth curve, the CT value is 26.8, and the detection sensitivity of the present invention can reach 10% of the K103N mutation sample.

(2) According to (1), use the same method to prepare 1%-100% V108I mutation samples, use primers SEQ ID NO.2 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 29.39, and the detection sensitivity of the present invention can reach 10% of mutant samples.

(3) According to (1), use the same method to prepare 1%-100% E138A mutation samples, use primers SEQ ID NO.3 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 28.2, and the detection sensitivity of the present invention can reach 5% of E138A mutation samples.

(4) According to (1), use the same method to prepare 1%-100% Y181C mutation samples, use primers SEQ ID NO.4 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 29.3, and the detection sensitivity of the present invention can reach 5% of Y181C mutation samples.

(5) According to (1), use the same method to prepare 1%-100% Y188C mutation samples, use primers SEQ ID NO.5 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 29.3, and the detection sensitivity of the present invention can reach 5% of Y188C mutation samples.

(6) According to (1), use the same method to prepare 1%-100% G190A mutation samples, use primers SEQ ID NO.6 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 28.9, and the detection sensitivity of the present invention can reach 5% of G190A mutation samples.

(7) According to (1), use the same method to prepare 1%-100% G190R mutation samples, use primers SEQ ID NO.7 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 28.6, and the detection sensitivity of the present invention can reach 5% of G190R mutation samples.

(8) According to (1), use the same method to prepare 1%-100% M230I mutation samples, use primers SEQ ID NO.8 and SEQ ID NO.9 as primers, respectively use 1% mutation samples, 5% mutation samples, 10% mutant samples and 100% mutant samples were used as templates for real-time fluorescent PCR reaction. The reaction system and reaction procedure were the same as (1). The value is 29.4, and the detection sensitivity of the present invention can reach 10% of M230I mutation samples.

Claims (2)

1. A primer for detecting the main drug-resistant mutation sites of non-nucleoside reverse transcriptase inhibitors for the treatment of AIDS, including detecting the mutation sites K103N, V108I, E138A, Y181C, Y188C, and G190A of HIV-1 virus pol gene , G190R and M230I allele-specific PCR primers, each mutation site includes a specific upstream primer and a general downstream primer, wherein, The upstream primer and downstream primer of the K103N site are the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the V108I site are the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the E138A site are the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the Y181C site are the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the Y188C site are the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the G190A site are the nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the G190R site are the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.9 respectively; The upstream primer and downstream primer of the M230I site are the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. 2. the application of the primer described in claim 1 in the detection of AIDS treatment drug non-nucleoside reverse transcriptase inhibitor resistance mutation site, the drug resistance mutation site is K103N, V108I, E138A, Y181C, Y188C, G190A , G190R and M230I.