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Preparation and method of lamifuding drug resistant gene chip for hepatitis virus B type

A hepatitis B virus, lamivudine technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to detect mutations, cumbersome operations, etc., to achieve specific detection, major social and economical problems effect of benefit

Inactive Publication Date: 2005-06-15
深圳益生堂生物企业有限公司
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AI Technical Summary

Problems solved by technology

The above methods either cannot detect all mutations, or the operation is cumbersome

Method used

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  • Preparation and method of lamifuding drug resistant gene chip for hepatitis virus B type
  • Preparation and method of lamifuding drug resistant gene chip for hepatitis virus B type
  • Preparation and method of lamifuding drug resistant gene chip for hepatitis virus B type

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0069] Example: oligonucleotide chip detection of hepatitis B virus lamivudine resistance gene mutation

[0070] 1. PCR amplification and fluorescent labeling of detection fragments

[0071] 1.1 HBV DNA extraction:

[0072] Take 15ul of serum to be tested and put it in 30ul of lysate, mix well, put in boiling water for 10 minutes, then centrifuge at 13000rmp for 10 minutes, take 2ul of supernatant as PCR template.

[0073] 1.2 PCR amplification reaction:

[0074] Take a sterilized centrifuge tube, prepare PCR reaction mixture (27.5ul PCR reaction solution × number of reaction tubes + 0.5ul enzyme mixture × number of reaction tubes), mix well, take 28μl PCR reaction mixture into each amplification tube, carefully Pipette 2 μl of the treated specimen supernatant into a PCR reaction tube, mix well, centrifuge at 8,000 rpm for a few seconds, treat at 37°C for 10 minutes, and then carry out PCR reaction according to the following parameters: 94°C for 5 minutes; 94°C for 0.5 minut...

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Abstract

A gene chip for detecting the resistance of hepatitis B virus to lamivudine has a thin glass substrate with multiple reaction areas. Each reaction area has a detection microarray consisting of 4 subarrays. Each subarrray has 30 probes composed of 28 probes for detecting the DNA polymerases of HBV, a HBV DNA probe and a negative reference probe. Its preparing process includes preparing relative reagents, preparing gene chip, and providing various kinds of solution. Its detection method includes PCR amplification, marking, hybridizing with said probes, washing, laser scanning to obtain images, converting image to digital values, and data processing.

Description

technical field [0001] The invention relates to a method for detecting hepatitis B virus lamivudine drug-resistant mutation by using a gene chip Background technique [0002] Hepatitis B virus infection is an important pathogenic factor of acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HBV infection is also one of the top ten leading causes of death worldwide. The total number of chronic hepatitis B patients in the world is about 400 million (Lok, A.S.J. Hepatol. 2000, 32: 89-97). Interferon-alpha and lamivudine are two commonly used drugs for the treatment of chronic hepatitis B. However, the effect of interferon therapy is not very satisfactory, and injections are required, and there are often some side effects. Lamivudine is a nucleoside analogue, which is administered orally, and is convenient to use, has rapid onset of effects, and little side effects (Jarvis, B., and D.Faulds.Drugs1999, 58: 101-141; Wong, D.K., A.M. Cheung, et al. Ann. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈立炎张文耿永尧尹忠华
Owner 深圳益生堂生物企业有限公司
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