HBV DNA gene subtype detecting method and kit

A technology for genotyping and detection methods, applied in the field of molecular biology, can solve the problems of cumbersome and complicated HBV DNA typing and detection, improve biosafety and detection stability, simplify settings and protection requirements, and avoid template contamination. Effect

Inactive Publication Date: 2007-10-03
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 technical problem to be solved in the present invention is to provide a detection method for HBV DNA typing and a test kit thereof, so as to overcome the cumbersome and complicated defects of the prior art in detecting HBV DNA typing

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Design and prepare primers and probe sequences. (Designed for HBV DNA gene-related sequences, GeneBank sequence numbers are Type B AB033555, Type C AB014377, Type D AB104709)

[0067] Primer 1 (upstream primer) 5'AGACT CGTGG TGGAC TTC 3',

[0068] Primer 2 (downstream primer) 5'AGGCA TAGCA GCAGG ATG 3',

[0069] Sequence B 5'AAATC TCCAG 3';

[0070] Sequence C 5'AGCAC CCACG 3';

[0071] Sequence D 5'ACTAC CGTGT 3';

[0072] Probe 1 (B genotype probe) 5'AGT CCC AAA TCT CCA G TC AC 3' (contains sequence B),

[0073] Probe 2 (C genotype probe) 5'GG A GCA CCC ACG TGT CCT GG 3' (contains sequence C),

[0074] Probe 3 (D genotype probe) 5'GA A CTA CCG TGT GTC TTG GC 3' (contains sequence D)

[0075] The fluorescent labels of probes 1, 2, and 3 are: the fluorescent luminescent group FAM; the fluorescent quenching group is TAMRA.

Embodiment 2

[0077] Prepare the hepatitis B virus (HBV) typing fluorescent PCR detection kit.

[0078] Composed of:

[0079] Composition (10 servings / box) Volume

[0080] Extract solution A 500μL×1 tube

[0081] Extract B 500μL×1 tube

[0082] Reaction solution 560μL×1 tube

[0083] Fluorescent probe B 60μL×1 tube

[0084] Fluorescent probe C 60μL×1 tube

[0085] MgCl 2 Solution 160μL×1 tube

[0086] Taq enzyme (containing 0.67U / μL Taq enzyme and 0.02U / LUNG enzyme) 90μL×1 tube

[0087] Positive control substance 20μL×1 tube

[0088] Negative control substance 50μL×1 tube

[0089] raw material

B reaction tube

C reaction tube

10×Buffer

1×

1×

MgCl 2

4mM

4mM

d(AGC)TP

200μM

200μM

d(UTP)

300μM

300μM

Primer 1, 2

200nM

200nM

Probe 1

300nM

Probe 2

300nM

Taq enzyme

2U

2U

UNGase

0.06U

0.06U

Template (additional when...

Embodiment 3

[0095] The typing detection method of HBV DNA is also the method of using the detection kit.

[0096] 1. HBV DNA extraction

[0097] Take 50 μL of the serum sample to be tested and add 50 μL of nucleic acid extraction solution A. Shake and mix for 15s. Centrifuge at 13,000rpm for 10min, discard the supernatant. Add 50 μL of well-mixed nucleic acid extraction solution B, shake and mix, bathe in 100°C water for 10 minutes, and centrifuge at 13,000 rpm for 3 minutes. Take the supernatant for PCR amplification.

[0098] 2. Fluorescent PCR detection

[0099] Prepare two tubes of reaction solution according to the number of samples [number of samples = number of samples + reference substance] n:

[0100] : Take the reaction solution n×28μL, MgCl 2 Mix n×8μL, fluorescent probe B n×6μL, Taq enzyme n×3μL in a centrifuge tube and mix well

[0101] : Take the reaction solution n×28μL, MgCl 2 Mix n×8μL, fluorescent probe C n×6μL, Taq enzyme n×3μL in a centrifuge tube and mix wel...

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PUM

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Abstract

The present invention relates to HBV DNA gene subtype detecting method and kit. Specific probe and matched primer or specific primer and matched reverse primer and specific virus detecting probe are designed based on HBV genotype characteristic sites for detecting HBV genotype alone or performing real-time fluoroscopic examination on the identical sample in several PCR tubes to detect type B and type C or general type. Through the detection, Ct difference is detected and the middle virus content in clinical HBV sample is judged. The kit of the present invention has stable performance, simple operation, fast detection and capacity of fast judging HBV DNA type, and is used in the HBV antagonizing treatment and prognosis analysis.

Description

technical field [0001] The invention relates to molecular biology techniques, in particular to the detection of a virus. 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 including the host and the virus. Viral genetic heterogeneity affects antigen expression and may also play an important role. Different strain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 夏懿沈维祥廖兴中吴大治
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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