Displacement amplification method for detecting hepatitis B viruse P gene YMDD variation

A hepatitis B virus, displacement amplification technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, fermentation, etc. Effect

Inactive Publication Date: 2008-08-27
北京万达因生物医学技术有限责任公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the deficiencies in the above fields, a displacement amplification method for detecting the variation of the hepatitis B virus P gene YMDD is provided, and the amplification sensitivity is reduced b

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  • Displacement amplification method for detecting hepatitis B viruse P gene YMDD variation
  • Displacement amplification method for detecting hepatitis B viruse P gene YMDD variation

Examples

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Embodiment Construction

[0038] The present invention will be described in further detail below in conjunction with embodiment. All reagents used are commercially available.

[0039] Example steps:

[0040] Serum samples from 6 cases of chronic hepatitis B infection and 1 case of normal person treated with long-term lamivudine. Extract with equal amount of phenol / chloroform and chloroform alone, and purify with DNA purification column from Tiangen Company. Add mixed indicator DNA molecule 1, molecule 2, molecule 3 and double-stranded oligomeric DNA inhibitor to each sample respectively, and the sample indicator DNA hybridization ligase reaction, using 1-2ul of the ligase reaction solution as a template, add reverse PCR Primers Reverse F and Reverse R, reverse PCR amplification. At the same time, a mixture of pUC YMDD (531-582aa), pUC YIDD and pUC YVDD plasmids was used as a positive control, and a single pUC19 plasmid was used as a negative control.

[0041] (1) Instructions for adding head and ta...

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Abstract

The invention relates to a displacement amplification process which is used to detect hepatitis b virus P gene YMDD variation, which is characterized in that detected HBV P gene YMDD motif and variation YIDD, wherein YVDD sequence is displaced into indication DNA through hybridization-ligase reaction, and then is reversely amplified into an induction system. The YMDD hybrid sequence is separated into left half and right half and is added on the front end and the tail end of a section of induction DNA, is hybridized with P gene YMDD region sequence after added with front and tail induction DNA, make the front end and tail end are in accordance with each other, and the hybrid gap is connected with ligase. The reverse amplification induction DNA adopts right middle sequence of induction DNA as primer, reversely amplifies the covalence connection of the front end and the tail end to induct DNA, wherein base groups on the two sides of the gap are connected through matching with dependency, thereby being suitable to one nucleotide mutagenic gene analysis. Detection can be displaced into several inducted PCR, thereby avoiding the recontamination of one set of system amplification products. Detected RNA can be displaced into induction DNA to be directly detected through hybridization-connection reaction, or is suitable to the real-time quantitative analysis of detected gene through combining with SYBR Green dye or fluorescent molecular probe.

Description

Technical field: [0001] The invention relates to a molecular detection method of the hepatitis B virus (Hepatitis B Virus HBV) gene and its variation. Especially HBV genome includes DNA and RNA and YMDD gene mutation polymerase chain reaction (Polymerase Chain Reaction) PCR amplification detection method and detection kit. Background technique: [0002] Hepatitis B is an infectious disease that seriously threatens human health. According to the report of the World Health Organization, about 2 billion people in the world have been infected with HBV, of which more than 350 million people are chronic hepatitis B virus HBV infected people. my country is a high prevalence area of ​​hepatitis B infection . Hepatitis B virus belongs to the hepadnaviridae family and is also the main cause of chronic hepatitis, cirrhosis, liver failure and primary hepatocellular carcinoma. The diagnosis of hepatitis B infection is currently mainly based on serological enzyme immunoassay detection. W...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 江洪廖同兵
Owner 北京万达因生物医学技术有限责任公司
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