Method for detecting A1762T-G11764A double-mutation of hepatitis B virus DNA and kit

A technology of DNA A1762T-G1764A and A1762T-G1764A, which is applied in the field of detection of hepatitis B virus DNA A1762T-G1764A double mutation, can solve the problems of troublesome sequencing methods, linear probe specificity is not as good as molecular beacons, etc., and achieve reliable detection results , The detection result is beautiful and stable, and the effect of reducing the detection cost

Active Publication Date: 2010-12-29
INTEC PROD INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequencing method is cumbersome, and the sensitivity of mutation detection is not as high as that of real-time fluorescent PCR; and the specificity of linear probes is not as good as that of molecular beacons (Marras S.A.E, Kramer F.R., Tyagi S. The Humana Press Inc. 2003; 212 :111-128)

Method used

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  • Method for detecting A1762T-G11764A double-mutation of hepatitis B virus DNA and kit
  • Method for detecting A1762T-G11764A double-mutation of hepatitis B virus DNA and kit
  • Method for detecting A1762T-G11764A double-mutation of hepatitis B virus DNA and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1: Extraction of hepatitis B virus DNA in clinical blood samples

[0079] 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

[0080] Embodiment 2: PCR amplification of target nucleic acid

[0081] 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 PCR machine (a PCR machine that can detect FAM), 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, 58°C °C for 30 seconds, 35 °C for 30 seconds (40 cycles). Among them, 58°C is the annealing temperature, and 35°C is the temperature for detecting fluorescence.

[0082] The PCR amplification system used included: 2.5 μl 10×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), two forward and reverse primers (SEQ ID NO: 1 and SEQ ID NO: 2, 100 ...

Embodiment 3

[0083] Example 3: Sensitivity and specificity detection of hepatitis B virus DNA A1762T-G1764A double mutation

[0084] The plasmid (1.0×10 6 copies / ml-1.0×10 2 copy / ml) is template, use reagent of the present invention to hepatitis B virus DNA A 1762 T-G 1764 The sensitivity of the A double mutation was detected, and the detection results can be found in the appendix 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 1.0×10 2 copy / ml of A 1762 T-G 1764 A double mutant hepatitis B virus DNA. In other words, the sensitivity of the present invention can fully meet the clinical needs.

[0085] With 10 plasmids (about 1.0×10 6 copies / ml) is template, use reagent of the present invention to hepatitis B virus DNAA 1762 T-G 1764 The specificity of the A double mutation was tested, and the test results can be found in the appendix figure 2 . from figure 2 I...

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Abstract

The invention relates to a double mutation detection method and a reagent box for a hepatitis B virus DNA A1762T-G1764A, in particular to a double mutation detection method for detecting the A1762T-G1764A, in the DNA sequence of the hepatitis B virus by utilizing a molecular beacon probe technology and a real time PCR method as well as a reagent box for finishing the detection. The method of the invention can specially and sensitively detect the double mutation hepatitis B virus DNA of the A1762T-G1764A in a clinic blood sample.

Description

field of invention [0001] The invention relates to the detection of hepatitis B virus DNA A 1762 T-G 1764 A double mutation method, in particular a real-time PCR detection method using molecular beacon fluorescent probe technology. 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 development of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). [0...

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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