Method for detecting hepatitis B virus DNA and G1896A mutation thereof and kit

A hepatitis B virus, G1896A technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of prolonging the running time of PCR programs, etc., achieve beautiful and stable amplification curves, reliable detection results, and benefit Promoted app performance

Active Publication Date: 2011-08-31
INTEC PROD INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This virtually prolongs the running time of the entire PCR program

Method used

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  • Method for detecting hepatitis B virus DNA and G1896A mutation thereof and kit
  • Method for detecting hepatitis B virus DNA and G1896A mutation thereof and kit
  • Method for detecting hepatitis B virus DNA and G1896A mutation thereof and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1: Extraction of hepatitis B virus DNA in clinical blood samples

[0098] Take 50 μl of serum from clinical blood samples into a high-pressure 1.5ml centrifuge tube, add 25 μl of extraction solution A (40% PEG-8000), shake and mix for 5 seconds, centrifuge at 12,500 g for 5 minutes; discard the supernatant, add 25 μl of extraction Solution B (0.25M NaOH), shake for 15s to dissolve the precipitate as much as possible, lyse at 96°C for 10min; add 25μl of extract solution C (0.4M Tris-HCl buffer, pH6.4), mix well, 96°C for 10min; centrifuge at 12,500g After 15 minutes, 5 μl of the supernatant was taken for PCR amplification.

Embodiment 2

[0099] Embodiment 2: PCR amplification of target nucleic acid

[0100] Add a single serving of PCR reaction mixture into a PCR reaction tube, then add 0.2 μl of enzyme mixture, and finally add 5 μl of template (template to be tested or negative and positive control), mix thoroughly and centrifuge slightly (about 5 Second). Then put the reaction tube into a fluorescent PCR instrument (a PCR instrument that can detect FAM and HEX), and amplify according to the following conditions: 50°C for 120 seconds (1 cycle) → 95°C for 180 seconds (1 cycle) → 95°C for 10 seconds seconds, 56°C for 30 seconds (40 cycles), where 56°C is the temperature for detecting fluorescence.

[0101] The PCR amplification system used includes: 2.5 μl 10x PCR buffer (850 mM KCl, 400 mM Tris-Cl, pH8.9, 50% v / v glycerol), 0.2 μl enzyme mixture, 0.2 μl dUTPs (containing dATP, dUTP, dGTP , dCTP each 25mM), forward and reverse two primers (SEQ ID NO: 1 and SEQ ID NO: 2, 100 μ M) each 0.1 μ l, wild-type and mut...

Embodiment 3

[0102] Example 3: Hepatitis B virus DNA G 1896 Sensitive and specific detection of A mutations

[0103] with G 1896 A plasmid (1.0x10) of a mutated hepatitis B virus DNA fragment 6 copies / ml-1.0x10 2 copies / ml) is template, use reagent of the present invention to hepatitis B disease

[0104] Toxic DNAG 1896 The sensitivity of A mutation is detected, and the detection results can be found in figure 1 . from figure 1 As can be seen from the results, the method and kit of the present invention can at least detect that the template concentration is only 1.0x10 2 copies / ml of G 1896 A mutated hepatitis B virus DNA. In other words, the sensitivity of the present invention can fully meet the clinical needs.

[0105] Then wild-type hepatitis B virus DNA (about 1.0×10 6 copies / ml) is a template, and the specificity of the FAM-labeled mutant probe is detected using the reagent of the present invention, and the detection results can be found in figure 2 . from figure 2 ...

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Abstract

The invention relates to a detecting method and a reagent box for the DNA of the hepatitis B viruses of wild type and G1896A mutant, in particular to a method for detecting the DNA of the hepatitis B viruses of wild type and G1896A mutant by using the technology of combining the Locked Nucleic Acids with a molecular beacon probe as well as a real time PCR method and a reagent box used for finishing the detection. The method of the invention can specially and sensitively detect the DNA of the hepatitis B viruses of wild type and G1896A mutant in a clinic blood sample.

Description

field of invention [0001] The present invention relates to the detection of wild type and G 1896 A method for mutant hepatitis B virus DNA, particularly relates to a method for detecting real-time PCR using a technology combining locked nucleic acid (LNA, Locked Nucleic Acids) and molecular beacon probes. The present invention further relates to a kit for the clinical detection. Background of the invention [0002] Viral hepatitis B (HB, hepatitis B) is a worldwide infectious disease (Lok AS, Heathcote EJ, Hoofnagle JH. Gastroenterology 2001; 120: 1828-1853). my country is a high-incidence area of ​​HBV infection, about 50-70% of the population has been infected with HBV, and about 8-12% of the population is chronic HBV infected. At present, about 1.2 million people die from diseases caused by HBV infection every year in the world. Hepatitis B virus infection can not only cause acute and chronic viral hepatitis, but also has a close relationship with the occurrence and de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 蔡剑英宋庆涛
Owner INTEC PROD INC
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