Clinical Significance And Method Of Detection HBV CccDNA In Liver Tissue By Novel In Situ Polymerase Chain Reaction

Hepatitis B virus (HBV) infection is a serious health problem in recent year that infection about350million people world wide,chronic HBV infection is associated with development of liver cirrhosis,liver failure and hepatocellular carcinoma, about have a15to25%chance of premature death due to cirrhosis or hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family, which is characterize btropism for the liver; a double-stranded, relaxed-circular (RCDNA genome; and DNA replication that involves reverse transcription of an RNA intermediate.The formation of covalently closed circular DNA (cccDNA) is crucial procedure in the HBV life cycles,which serves as original template of PgRNA(pregene RNA) for viral replication and play important role in the persistence of HBV infection. Detection of HBV cccDNA distribution in the liver is suggested to be valuable for research pathogenic mechainism of HBV and estimate anti-HBV therapeutic effect. We developed a novel PCR method for detecting and visualizing HBV cccDNA. which was capable of detecting samples as low as102copies of serum HBVDNA loading.Objective To develop a sensitive and specific for detecting HBV cccDNA in liver tissue section obtained from the patients with chronic hepatitis B(CHB) liver cirrhosis,liver failure and hepatocellular carcinoma patients by a novel situ polymerase chain reaction and to explore its clinical significance.Method With Chronic hepatitis B, liver cirrohsis and hepatoceular carcinoma tissue samples were obtained by biopsy and autopsy in30cases, Each sample was detected for HBsAg and HBcAg by immunohistochemistry expression for first and then. Each sample was detected for HBV cccDNA by a novel PCR,the liver tissue sections were treated by Plasmid-safe ATP-dependent DNase (PSAD) was used prior to in situ PCR, then use in situ rolling circle amplification (RCA) to amplify HBV cccDNA, A pair of digoxigenin labeled HBV cccDNA-selective primers spanning across the gap region of the HBV genome were used by in situ polymerase chain reaction, after in situ PCR, the signals were detected by immunohistochemistry methods, results compare with in semi-situ polymerase chain reaction and analyze sensitivity and specificity of the novel in situ PCR.Results In all30cases detected by novel situ PCR,20showed HBV cccDNA positive (67%). HBV cccDNA was distributed in chronic type B hepatitis with an expression rate of35%-70%. Positive signal presented as purple blue, blue or purple block or granules located in affected hepatocyte nucleus. No signal was appeared in negative controls. Compare with in semi-situ polymerase chain reaction, distribution of HBV cccDNA is more concentrated in novel situ PCR, the serum HBVDNA is more than106Copies/ml with a higher detection rate, novel in situ PCR method for detecting and visualizing HBV cccDNA was capable of detecting as low as102copies of serum HBVDNA loading of samples, HBcAg positive patients with a higher detection rate.Our results indicate that in situ detection of HBV cccDNA with a highly sensitive method is important as well as serology.Conclusions Novel in situ polymerase chain reaction is a potential practicable method for specific and efficient quantification of HBV cccDNA and the primary positive signal is in the nucleus. It indicates that HBV cccDNA is mainly located in liver nucleus which is the source of persistent viral infection and replication of HBV.