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DNA chip for detecting main drug-resistant mutation of HIV reverse transcriptase regions, preparation thereof and application thereof

A technology of reverse transcriptase region and DNA chip, which is applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., and can solve problems such as HIV drug-resistant mutations

Pending Publication Date: 2019-01-11
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expansion of antiviral therapy has also promoted the occurrence of HIV drug-resistant mutations and drug-resistant strains

Method used

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  • DNA chip for detecting main drug-resistant mutation of HIV reverse transcriptase regions, preparation thereof and application thereof
  • DNA chip for detecting main drug-resistant mutation of HIV reverse transcriptase regions, preparation thereof and application thereof
  • DNA chip for detecting main drug-resistant mutation of HIV reverse transcriptase regions, preparation thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, design and preparation of biochip

[0027] (1) Primer and probe design

[0028] 16 drug-resistant mutation sites in the HIV reverse transcriptase region were used as detection genes, and the 16 common drug-resistant sites were: K70E, K103N, K65R, K70R, V108I, Y188C, Y181C, M184V, M184I, L210W, M230I, G190R . Designed according to the principle of allele-specific detection, the primers include a universal downstream primer and multiple specific upstream primers (18-27bp in length), in which the 3' terminal base of the wild-type specific upstream primer at the drug-resistant site is the same as the detected site The wild-type site sequence of the point is completely complementary, and the 3' terminal base of the mutant-type specific upstream primer is completely complementary to the mutant-type site sequence of the detected site, so when performing PCR detection, the specific upstream primer only amplifies the the complementary sequence. A general downstr...

Embodiment 2

[0043] Example 2, the application of biochips in the detection of drug-resistant mutations in the HIV reverse transcriptase region

[0044] (1) PCR amplification of the sample to be tested

[0045] The DNA or cDNA of the sample to be detected, the cy3-modified probe-primer chain, and the universal downstream primer are used for PCR reaction in the same tube.

[0046]The amplification template of conventional PCR amplification method is cDNA obtained by reverse transcription of RNA extracted from HIV patient plasma or DNA extracted from artificially constructed plasmid. The artificially constructed plasmids in this example are the recombinant plasmids containing the HIV reverse transcriptase region gene constructed with the pUCm-19 vector in the previous laboratory, including wild-type plasmids without drug-resistant mutations and mutant plasmids containing the above-mentioned drug-resistant mutations: Plasmid-HIV-WT, Plasmid-K70E, Plasmid-K103N, Plasmid-K65R, Plasmid-K70R, Pl...

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Abstract

The invention discloses a DNA chip for detecting main drug-resistant mutation of HIV reverse transcriptase regions, comprising 40 oligonucleotide probes of main drug-resistant mutation of 16 HIV reverse transcriptase region, and the oligonucleotide probes distributed on a carrier to form a lattice. At the same time, the inventors also established a preparation method of the corresponding DNA chip.The DNA chip prepared by the invention can simultaneously detect the main drug-resistant mutations of 16 common HIV reverse transcriptase regions. As proved by experiments, that invention detect thedrug resistance site of the reverse transcriptase region of the HIV virus to be detected, the result is intuitive and accurate, the complicated analysis process of the DNA direct sequence method is avoided, and the invention has a guiding effect on the determination of the AIDS antiviral treatment scheme, and accordingly, a corresponding detecting kit can be prepared and used for the AIDS drug resistance monitoring. In short, the DNA chip has the characteristics of quick, sensitive, efficient and lower cost, which provides a new detecting method for HIV drug resistance epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of gene chip detection, and in particular relates to a DNA chip for detecting the main drug-resistant mutation in the HIV reverse transcriptase region and its preparation and application. Background technique [0002] In 2003, my country began to implement highly active antiretroviral therapy (HARRT) for AIDS patients. This method can effectively inhibit the replication of AIDS virus (HIV) in AIDS patients, reduce the mortality rate and prolong the life of patients. However, the expansion of antiviral therapy has also promoted the occurrence of HIV drug-resistant mutations and drug-resistant strains. HIV drug-resistant mutations are divided into drug-resistant mutations in the protease region and drug-resistant mutations in the reverse transcriptase region. There are nearly 40 major drug-resistant mutations in the reverse transcriptase region. The detection of drug-resistant strains can not only grasp the e...

Claims

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Application Information

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IPC IPC(8): C12Q1/6837C12Q1/6827
CPCC12Q1/6827C12Q1/6837C12Q2535/131C12Q2565/501
Inventor 梁浩叶力陈荣凤蒋俊俊臧宁梁冰玉余军黄颉刚宁传艺廖艳研刘洁
Owner GUANGXI MEDICAL UNIVERSITY
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