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Kit for detecting hepatitis B virus cccDNA (Deoxyribonucleic Acid) through fluorescent quantification PCR (Polymerase Chain Reaction) of rolling cycle augmentation spanned notch

A technology for quantitative detection of hepatitis B virus, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc. Closed circular DNA and other problems, to achieve specificity and sensitivity improvement, increase the rolling circle amplification link, good specificity effect

Inactive Publication Date: 2011-01-19
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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Problems solved by technology

Because the content of HBV cccDNA is usually much lower than that of HBV relaxed circular DNA (rcDNA), it is difficult to ensure the specificity and sensitivity of detection by real-time fluorescent PCR method and Invader method across the gap
[0008] 2. Invader technology and cross-gap real-time fluorescent quantitative PCR technology cannot distinguish integrated DNA and cccDNA
[0009] There is a large amount of integrated HBV DNA in the liver tissue of chronic HBV infection, especially in patients with liver cirrhosis. Even if the current advanced PSAD enzyme is used to digest non-closed circular DNA, it still cannot guarantee that the non-closed circular DNA is completely digested, and it cannot effectively distinguish cccDNA from cccDNA. integrated HBV DNA
[0010] 3. Inability to effectively distinguish integrated DNA, relaxed circular DNA (rcDNA) and closed circular DNA (cccDNA)
[0011] 4. Differences between samples interfere with data analysis, and there is no internal reference for standardization
[0014] Rolling circle amplification (RCA) is characterized by the fact that it can only amplify closed circular DNA and cannot effectively remove non-closed circular DNA that has not been fully digested by PSAD enzyme, so it is difficult to complete the quantitative detection of HBV cccDNA only by rolling circle amplification

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  • Kit for detecting hepatitis B virus cccDNA (Deoxyribonucleic Acid) through fluorescent quantification PCR (Polymerase Chain Reaction) of rolling cycle augmentation spanned notch
  • Kit for detecting hepatitis B virus cccDNA (Deoxyribonucleic Acid) through fluorescent quantification PCR (Polymerase Chain Reaction) of rolling cycle augmentation spanned notch
  • Kit for detecting hepatitis B virus cccDNA (Deoxyribonucleic Acid) through fluorescent quantification PCR (Polymerase Chain Reaction) of rolling cycle augmentation spanned notch

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Embodiment 1

[0051] Example 1 Rolling circle amplification increases real-time fluorescence quantitative PCR across the gap to detect cccDNA in liver tissue of patients with hepatitis B

[0052] 1. Research samples

[0053] The subjects of the study were 140 patients with chronic hepatitis B from May 2008 to May 2009 in the 302 Hospital of the People's Liberation Army, including 93 males and 47 females, ranging in age from 3 to 68 years old. The diagnosis was in line with the diagnostic criteria for viral hepatitis revised by the Tenth National Viral Hepatitis Conference in 2000, excluding hepatitis A, C, D, E, and G viruses, transfusion-transmitted viruses, cytomegalovirus, Epstein-Barr virus infection, and alcoholic and drug-related infections. Sexual, autoimmune liver disease. Liver tissue samples from non-hepatitis B patients were also collected as negative controls.

[0054] 2. Method

[0055] 1. Extraction of total DNA from liver tissue:

[0056] 5 mg of fresh frozen liver tissue...

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Abstract

The invention relates to a method for quantitatively detecting hepatitis B virus cccDNA (Deoxyribonucleic Acid) by adopting a real-time fluorescent PCR (Polymerase Chain Reaction) technology of a rolling cycle augmentation spanned notch, and a novel detection kit developed by the method. The invention can simultaneously improve the specificity and the sensitivity of a detection reaction.

Description

Technical field: [0001] The present invention relates to a method for the quantitative detection of hepatitis B virus cccDNA, in particular to a method for the quantitative detection of hepatitis B virus covalently closed circular DNA by using rolling circle amplification to increase cross-gap fluorescent quantitative PCR technology; Methods The hepatitis B virus cccDNA detection kit was developed. Background technique: [0002] Hepatitis B virus covalently closed circular DNA (covalently closed circular DNA, hereinafter referred to as HBV cccDNA) is replicated in the cell after hepatitis B virus infects the host cell and establishes an infectious state, and the virus relaxed circular DNA (relaxed circular DNA, rcDNA) Enter the nucleus, and use host DNA polymerase and topozyme to "repair" the formed structurally complete, supercoiled double-stranded DNA molecule. [0003] At present, the main method of HBV cccDNA detection is fluorescent probe detection technology. Through ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 钟彦伟徐东平韩佳琪苏何玲任晓强
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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