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Nest type-real time quantitative PCR method for detecting hepatitis B virus cccDNA

A hepatitis B virus, real-time quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, etc. Sensitivity, wide application range, accurate results of operation process and result analysis

Inactive Publication Date: 2008-01-16
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] It can be seen that although the above methods have their own advantages and disadvantages, there are still some problems in clinical application, such as low sensitivity and specificity, complicated operation, high cost, and limited detection of liver tissue samples.

Method used

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  • Nest type-real time quantitative PCR method for detecting hepatitis B virus cccDNA

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Embodiment Construction

[0036] Below by example, the present invention is further explained.

[0037] 1. Specimen collection: extract 3ml of fresh whole blood from patients with hepatitis B, and separate serum; extract 5ml of fresh whole blood from patients with hepatitis B, separate PBMCs by density gradient centrifugation, and count cells; collect liver biopsy and liver resection specimens from patients with chronic hepatitis B, cirrhosis, and liver cancer. Freeze at -70°C.

[0038] 2. DNA extraction:

[0039] A. Serum viral DNA was extracted with QIAamp MinElute Virus Spin Kit from QIAGEN;

[0040] B. Plasmid kit to extract virus DNA in PBMC: Plasmid extraction kit (Beijing Tianwei Times Company) was used to extract virus DNA in PBMC.

[0041] C. DNA in liver tissue was extracted with genomic DNA extraction reagent (Beijing Tianwei Times Company).

[0042] The extracted DNA was dissolved in 50 μl TE or deionized water and frozen for future use.

[0043] 3. Design and synthesis of primers and p...

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Abstract

The invention relates to a nested quantitative PCR detection method for virus covalently closed circular DNA (ccc DNA) of hepatitis b virus. The particular detection steps are: 1) extraction DNA of hepatitis b virus; 2) design and synthesis primers and probe: design an internal and an external primer at conservative region of both sides of negative chain gap of HVB DNA, with Taqman fluorescence probe arranged at the lower part of the negative chain gap; 3) Plasmid-SafeTM ATP-Dependent DNase restriction enzyme purification; 4) nested real time quantitative PCR amplification, comprising: common PCR-template with external primer amplification purification and real time quantitative PCR-fluorescence quantitative amplification to common PCR production with internal primer and fluorescence probe. The invention can improve the specificity and sensitivity of detection reactive to make the operation process and result analysis much more accurate, simple and time saving.

Description

technical field [0001] The invention relates to a detection method for hepatitis B virus, in particular to a nested-quantitative PCR detection method for covalently closed circular DNA (cccDNA) of hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) seriously endangers human life and health. After it invades the liver cells, it removes the nucleocapsid in the cytoplasm to form a relaxed circular DNA (rcDNA), the two strands of which are not closed. When rcDNA enters the liver cell nucleus, it relies on the DNA polymerase of the host cell to fill the gaps on the two strands, and then folds and distorts cccDNA to form a superhelical structure, which is used as a template to transcribe into mRNAs of different sizes, and then translated into various viral protein. The 3.5Kb mRNA is used as a template for reverse transcription, using viral reverse transcriptase to synthesize full-length genomic negative-strand DNA, and then using negative-strand DNA as a tem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 白雪帆王平忠陈延平黄长形连建奇
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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