Method for detecting medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus

A drug-resistant mutation, hepatitis B virus technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problem of low sensitivity of YMDD mutation detection, inability to achieve mutant DNA quantification, and expensive detection costs and other problems, to achieve the effect of simplifying design ideas, shortening detection time, and low cost

Active Publication Date: 2011-11-02
THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

At present, there are many molecular biological detection methods for YMDD mutations. However, these methods are time-consuming in clinical practice, require high technical requirements, have low detection sensitivity for YMDD mutations, and are expensive to detect, and cannot quantify mutant DNA.

Method used

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  • Method for detecting medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus
  • Method for detecting medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus
  • Method for detecting medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus

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Embodiment 1

[0032] Sources of materials and reagents:

[0033] Escherichia coli DH5α Competent Cells Tiangen Biochemical (Beijing) Co., Ltd.

[0034] pMD 18-T Vector Bao Biological Engineering (Dalian) Co., Ltd.

[0035] Agarose Gel DNA Recovery Kit Beijing Biotech Biotechnology Co., Ltd.

[0036] High Purity Plasmid Miniprep Kit Beijing Biotech Biotechnology Co., Ltd.

[0037] 2×HotStart Taq PCR MasterMix Tiangen Biochemical (Beijing) Co., Ltd.

[0038] SYBR Premix Ex Taq TM Treasure Bioengineering (Dalian) Co., Ltd.

[0039]X-Gal, IPTG, DMF Shanghai Jierui Biological Engineering Co., Ltd.

[0040] 1. Design and synthesis of primers

[0041] 1.1 Principle of ARMS experiment

[0042] The main principle is to artificially create base mismatches between primers and templates, resulting in hindered amplification. The premise is that the key enzyme of PCR - heat-resistant Taq DNA polymerase lacks exo-cutting (3′→5′) proofreading activity, and this property is use...

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Abstract

The invention provides a method for detecting medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus, which detects the medicine-resistance mutation of tyrosine-methionineaspartate-aspartate (YMDD) of hepatitis B virus by using a real time-amplification refractory mutation system-quantitative polymerase chain reaction (RT-ARMS-q PCR) process. In the invention, sequence specific primers and a fluorescent dye SYBRGreenI are used for amplification, the real-time monitoring of a reaction is realized, the detection time is shortened, operation steps are simplified, an analysis process after amplification is not needed, and the possibility of product pollution is lowered; and compared with a Taqman probe requiring a double marking process, the SYBRGreenI does not need designing and synthesizing a fluorescent probe, the design concept is simplified, and the cost is low. And by combining fusion curve analysis, reaction specificity is realized.

Description

technical field [0001] The invention relates to a method for detecting drug resistance mutation of hepatitis B virus YMDD. Background technique [0002] Antiviral therapy is the key to the treatment of chronic hepatitis B. The ultimate goal is to suppress HBV replication to the greatest extent, reduce liver cirrhosis, hepatocellular carcinoma, and liver function decompensation, prolong survival, and improve quality of life. Nucleotide analogs can block the synthesis of HBV DNA and inhibit virus replication, while lamivudine is a nucleoside analog widely used in HBV treatment, which can significantly reduce the level of HBV DNA in serum, and can normalize ALT and Liver histology improved. However, long-term use of lamivudine for antiviral therapy is prone to mutations in the YMDD motif of HBV, leading to drug resistance, which is called YMDD mutation. The YMDD mutation makes the virus replication rebound in the patient, and the disease is aggravated. Therefore, early and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 欧启水商红艳
Owner THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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