Kit for detecting hepatitis B virus (HBV) cccDNA (Covalently Closed Circular DNA) of paraffin embedded hepatic tissue

Abstract

The invention relates to a kit for quantitatively detecting hepatitis B virus (HBV) cccDNA (Covalently Closed Circular DNA) of paraffin embedded hepatic tissue, which is characterized by comprising the following steps of: carrying out paraffin embedding on hepatic tissue; then respectively adding extracted total DNA into an HBV rolling loop primer and a notch crossing primer; and carrying out rolling and notch crossing fluorescent quantitative amplification. The kit has the advantages of no need of fresh hepatic tissue as a detecting sample, high detecting specificity and high sensitivity.

Description

Technical field: [0001] The invention relates to a method for quantitative detection of hepatitis B virus cccDNA, in particular to a method and a detection kit for quantitative detection of hepatitis B virus cccDNA in trace paraffin-embedded liver tissue. Background technique: [0002] Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is a key factor in HBV persistent infection and disease recurrence after antiviral drug withdrawal. Quantitative detection of HBV cccDNA is of great significance for evaluating the effect of antiviral treatment in patients with hepatitis B, but it has not been carried out in clinical practice. Two factors limit the practical application of HBV cccDNA testing. [0003] On the one hand, it is a question of the source of the sample: [0004] Since the cccDNA of hepatitis B virus mainly exists in the nucleus of liver cells, fresh human liver tissue should be used as a test sample, but fresh liver tissue is not easy to obtain. Unless ...

AI Technical Summary

Problems solved by technology

Since 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

[0014] 2. Inability to distinguish integrated DNA from cccDNA

[0015] There is a large amount of integrated HBV DNA in the liver tissue of patients with chronic HBV infection, especially cirrhosis. Even if the current advanced PSAD enzyme is used to degrade 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

[0016] 3. Inability to effectively distinguish integrated DNA, relaxed circular DNA (rcDNA) and closed circular DNA (cccDNA)

[0017] 4. Differences between samples interfere with data analysis, and there is no internal reference for standardization

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