Kit and detection method for detecting hepatitis B virus core gene promoter base mutation

A technology of hepatitis B virus and kit, applied in the field of molecular biology, can solve problems such as difficulty in popularization and application, too strict requirements on hybridization temperature, etc., and achieve high accuracy

Active Publication Date: 2013-08-07
CANVEST WUHAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (3) PCR-oligonucleotide probe hybridization: This method uses oligonucleotide fragments as probes and hybridizes under the premise of strictly controlling the hybridization conditions. The hybridization temperature requirements are too strict, so it is difficult to popularize and apply

Method used

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  • Kit and detection method for detecting hepatitis B virus core gene promoter base mutation
  • Kit and detection method for detecting hepatitis B virus core gene promoter base mutation
  • Kit and detection method for detecting hepatitis B virus core gene promoter base mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the detection reagent of group A of PCR of hepatitis B virus core gene promoter DNA in the kit, to the positive quality control material of application national regulation HBV standard substance, people's negative HBV serum, negative quality control substance and different dilution concentration, carry out negative And Positive Cutoff Test, Minimum Detection Concentration Test.

[0061] Both wild-type and mutant types of hepatitis B virus bases can be successfully amplified by PCR, including the following components:

[0062] Human negative HBV serum: no hepatitis B virus.

[0063] Negative quality control: No A1762T and Gl764A base variant wild-type gene clone strain and A1762T and G1764A base variant gene clone strain, wild-type gene clone strain (Escherichia coli DH5α) plasmid DNA, nucleotide sequence Yes:

[0064] gtttaaagac tgggaggagt tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctca...

Embodiment 2

[0088] Embodiment 2: A group of detection reagents of hepatitis B virus core gene promoter DNA in the kit can carry out PCR amplification smoothly to the A1762T mutant type of hepatitis B virus base, including the following components:

[0089] The negative quality control substance that detection test is used: with embodiment 1.

[0090] The positive quality control substance that detection test is used: with embodiment 1.

[0091] PCR reaction solution B: PCR buffer (1×), TaqDNA polymerase 1.5u, dNTP0.3μmol / L, and:

[0092] HBV-B1 upstream primer 5′-tgggg gagga gatta ggtta at-3′ 0.3 μmol / L

[0093] HBV-B2 upstream antisense primer 5′-tttaa cctaa tctcc tcccc ca-3′ 0.03 μmol / L

[0094] HBV-D common downstream primer 5′-agcca cccaa ggcac agctt g-3′; 0.3 μmol / L

[0095] HBV-Y fluorescent probe: 5′-FAM-tgtag gcata aattg gtctg tt-TAMARA-3′; 0.2μmol / L

[0096] Specific implementation steps:

[0097] 1) Sample DNA treatment: Take the negative quality control and positive qualit...

Embodiment 3

[0107] Embodiment 3: a group of detection reagents of hepatitis B virus core gene promoter DNA in the kit can carry out PCR amplification smoothly to the Gl764A mutant type of hepatitis B virus base, including the following components:

[0108] The negative quality control substance that detection test is used: with embodiment 1.

[0109] The positive quality control substance that detection test is used: with embodiment 1.

[0110] PCR reaction solution C: PCR buffer (1×), TaqDNA polymerase 1.5u, dNTP0.3μmol / L, and:

[0111] HBV-C1 upstream primer 5′-ggagg agatt aggtt aaaga-3′0.3μmol / L

[0112] HBV-C2 upstream antisense primer 5′-ccttt aacct aatct cctcc-3′0.03μmol / L

[0113] HBV-D common downstream primer 5′-agcca cccaa ggcac agctt g-3′0.3μmol / L

[0114] HBV-Y fluorescent probe: 5′-FAM-tgtag gcata aattg gtctg tt-TAMARA-3′0.2μmol / L

[0115] Specific implementation steps:

[0116] 1) Sample DNA treatment: Take the negative quality control and positive quality control, dilu...

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PUM

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Abstract

The invention discloses a kit and a detection method for detecting hepatitis B virus core gene promoter base mutation. The kit comprises a PCR reaction solution A, a PCR reaction solution B and a PCR reaction solution C. According to the present invention, the primer used by the method is designed mainly based on hepatitis B virus core gene promoter target gene and base variation thereof; competitive inhibition of the non-mutated (wild-type) reverse complementary sequence primer on the wild-type base sequence during PCR amplification is adopted to substantially prevent wild strain DNA amplification so as to accurately detect gene variation; and the kit has characteristics of convenient detection, stable result, low cost and high sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting the base mutation of the hepatitis B virus core gene promoter, and also to a detection method for the base mutation of the hepatitis B virus core gene promoter. Background technique [0002] Viral hepatitis B (HB, hePatitisB) is a worldwide infectious disease. Among the nearly 30 million chronic hepatitis B patients in China, most of the HBV mutant strains carried by them are expressed in front of the viral genome. The promoter base mutation of the core gene in the C region and the mutation in the HBV BCP region have a high incidence in patients with chronic severe hepatitis B, suggesting that the HBV BCP mutation is related to the pathogenesis and severity of hepatitis B. [0003] At present, the methods used to detect gene mutations at home and abroad include: [0004] (1) Direct PCR sequencing: direct sequence analysis of PCR products, and comparative anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 夏晓兵糜克永刘静占霖张易莎
Owner CANVEST WUHAN BIOTECH
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