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HBV DNA gene parting fluorescence PCR multicenter detecting method and kit thereof

A multi-channel detection and genotyping technology, which is applied in the field of fluorescent PCR multi-channel detection method and kit for hepatitis B virus genotyping, can solve the problems of cumbersome and complicated HBV genotyping detection, and improve biological safety The stability of sex and detection, the requirements for simplifying settings and protection, and the effect of avoiding template contamination

Inactive Publication Date: 2008-06-11
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The object of the present invention is to provide a kind of detection method of more effective and simple hepatitis B virus genotyping; Detect more cumbersome and complex defects

Method used

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  • HBV DNA gene parting fluorescence PCR multicenter detecting method and kit thereof
  • HBV DNA gene parting fluorescence PCR multicenter detecting method and kit thereof

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Embodiment approach

[0047] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the invention. After reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0048] For the experimental methods not indicated in the following examples, the specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

Embodiment 1

[0050] Design and prepare primers and probe sequences. (Designed for related sequences of HBV genome, GeneBank sequence number is B type AF121244, C type AB033553, D type AY741794)

[0051] Primer 1 (upstream primer) 5'-AGACT CGTGG TGGAC TTCTC-3'

[0052] Primer 2 (upstream primer) 5'-TGAGG CATAG CAGCA GGATG-3'

[0053] B Genotype Probe 5'CGCAG TCC CAAATC TCCA G TCAC-3' (comprising sequence B),

[0054] C genotype probe 5'-AGCACCCA CGTGTCCTGG CCAA-3' (comprising sequence C),

[0055] D genotype probe 5'-GCATG G GGCAGAATC T TTCC ACC-3' (contains sequence D)

[0056] The fluorescent label of the B genotype probe is: the fluorescent luminescent group FAM; the fluorescent quenching group is TAMRA.

[0057] The fluorescent label of the C genotype probe is: the fluorescent luminescent group JOE; the fluorescent quenching group is TAMRA.

[0058] The fluorescent label of the D genotype probe is: the fluorescent luminescent group Cy5; the fluorescent quenching group is BHQ-...

Embodiment 2

[0060] Prepare HBV genotyping real-time fluorescent PCR multi-channel detection kit.

[0061] The composition is (10 servings / box):

[0062]

[0063] Reaction solution formula in the kit:

[0064]

[0065] Nucleic acid extraction solution formula:

[0066] Extract A: 8% polyethylene glycol, 1M NaCl

[0067] Extract B: 10mM NaOH, 0.1% SDS, 15% Chelex-100, 1% Tween-20

[0068] The positive control substance is a mixture of artificially synthesized HBV type B, C and D DNA fragments.

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Abstract

The invention relates to a HBV-DNA gene typing fluorescence PCR multichannel detection method and the reagent box thereof. The method is characterized in that a HBV-DNA full sequence adopting the gene typing is found out of GenBank, and then sequence alignment is performed to the HBV-DNA full sequence; a gathering area of a type specific base of an HBV genotype is found out according to the sequence alignment result; a probe and the matched primer thereof are designed according to the gathering area of the type specific base, multichannel genotype detection and general type detection are performed in a PCR pipe through adopting a multicolour marking probe, and according to the difference value of the Ct value obtained through the multichannel genotype detection and general type detection, the proportion content of the HBV virus medium virus in the clinical samples is judged. The reagent box of the invention has the advantages that the property is stable, the operation is simple and convenient, the detection is quick, the type of the HBV-DNA can be perfectly judged, and the reagent box is in favor of judging the prognosis of chronic hepatitis B virus infected persons more comprehensively and choosing more appropriate therapeutic schedule.

Description

technical field [0001] The invention relates to molecular biology techniques, in particular to a fluorescent PCR multi-channel detection method for genotyping of hepatitis B virus (Hepatitis BVirus, HBV) and a kit thereof. Background technique [0002] Hepatitis B virus (HBV) is prevalent all over the world. It is estimated that there are 2 billion people infected in the world, of which 350 million are chronically infected, and my country accounts for about half of them. About 1 million people die of HBV infection in the world every year. This infection can eventually develop into liver cirrhosis and liver cancer, which is the ninth leading cause of death among human diseases announced by the WHO. [0003] Hepatitis B virus mainly causes liver damage through the host's immune mechanism. The body's cells recognize the virus antigen and attack the infected liver cells to cause inflammation. This process is affected by multiple factors of the host and the virus. Viral genetic ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 沈维祥廖兴中吴大治夏懿
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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