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

A hepatitis B virus, G1896A technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as prolonging the running time of PCR programs, achieve beautiful and stable amplification curves, reduce detection costs, and facilitate The effect of promoting the application

Active Publication Date: 2009-01-07
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

[0098] Example 1: Extraction of HBV DNA from clinical blood samples

[0099] Take 50μl of serum from clinical blood samples into a high-pressure 1.5ml centrifuge tube, add 25μl of extract A (40% PEG-8000), shake and mix for 5s, and centrifuge at 12,500g for 5min; discard the supernatant, add 25μl to extract 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 C (0.4M Tris-HCl buffer, pH6.4), mix well, 96°C for 10min; centrifuge at 12,500g After 15 min, 5 μl of supernatant was taken for PCR amplification.

Embodiment 2

[0100] Example 2: PCR Amplification of Target Nucleic Acids

[0101] Add a single dose of PCR reaction mixture to 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 well and then centrifuge slightly (about 5 μl). second). Then put the reaction tube into a fluorescent PCR machine (a PCR machine 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 at which fluorescence is detected.

[0102] The PCR amplification system used includes: 2.5μl 10x PCR buffer (850mM KCl, 400mM Tris-Cl, pH8.9, 50% v / v glycerol), 0.2μl enzyme mixture, 0.2μl dUTPs (containing dATP, dUTP, dGTP, dCTP each 25mM), two forward and reverse primers (SEQ ID NO: 1 and SEQ ID NO: 2, 100 μM) each 0.1 μl, wild type and mutant molecu...

Embodiment 3

[0103] Example 3: Hepatitis B virus DNA G 1896 Sensitive and specific detection of A mutation

[0104] with G 1896 A plasmid of mutated hepatitis B virus DNA fragment (1.0 × 10 6 copies / ml-1.0×10 2 copies / ml) as a template, use the reagent of the present invention to treat hepatitis B disease

[0105] Toxic DNA G 1896 The sensitivity of A mutation is detected. For the detection results, see figure 1 . from figure 1 It can be seen from the results that the method and kit of the present invention can at least detect that the template concentration is only 1.0×10 2 copies / ml G 1896 A mutated hepatitis B virus DNA. In other words, the sensitivity of the present invention can fully meet the clinical requirements.

[0106] Then 16 wild-type hepatitis B virus DNA (about 1.0 × 10) with no mutation at site 1896 6 copies / ml) is a template, and the specificity of the mutant probe marked by FAM is detected using the reagent of the present invention, and the detection results re...

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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 detection method using locked nucleic acid (LNA, Locked Nucleic Acids) and molecular beacon probe combination technology for real-time PCR. 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 deve...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 蔡剑英宋庆涛
Owner INTEC PROD INC
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