Establishment And Preliminary Application Of A Fluorescence-Based Quantitative PCR Assay For Detecting HBV CccDNA

Objective:To establish a new fluorescence-based quantitative PCR assay for detecting hepatitis B virus covalently closed circular DNA(HBV cccDNA).To explore the effect of artificial liver support system(ALSS)on total HBV DNA and HBV cccDNA in sera of patients with fulminant hepatitis B.Methods:Plasmids carrying HBV fragment were amplified with the specific primer set and fluorescent probe to establish the standard curve and to determine the sensitivity and reproducibility of the method.HBV DNA positive sera samples from patients with chronic hepatitis B(mild)were amplified simultaneously to test the specificity of the established fluorescence-based quantitative PCR method . Furthermore, the sera specimens from 50 patients of fulminant hepatitis B prior to and after treatment with artificial liver support system were collected.The genomic DNAs were extracted from sera of these patients and were further purified by Plasmid_SafeTMATP_dependent DNase to remove HBV relaxed circular DNA(rc DNA)and the other form of HBV DNA except HBV cccDNA.Then HBV cccDNA was quantitatively detected by fluorescent PCR with specific primer set and Taqman probe.And the results were analyzed by SAS8.0 statistics software.Results : We successfully established a new fluorescence-based quantitative PCR method for HBV cccDNA with the linear range from 1×102 to 2.77×109copies per milliliter.HBV cccDNA was not observed in HBV DNA positive sera sample from patients with chronic hepatitis B (mild).However, HBV cccDNA was detectable in sera of the patients with fulminant hepatitis B.Interestingly, The level of total HBV DNA and HBV cccDNA decreased significantly following plasma exchange in artificial liver support system.The levels of total HBV DNA and HBV cccDNA had positive correlation with degree of PT,ALT,AST.Total HBV DNA also had positive correlation with HBV cccDNA.Conclusion:This study established a fluorescence-based quantitative PCR method for detecting HBV cccDNA, with wide linear range,high sensitivity,specificity and reproducibility.HBV cccDNA existed in sera of patients with fulminant hepatitis B although was not detected in mild hepatitis B. The level of total HBV DNA and HBV cccDNA has positive correlation with degree of PT,ALT and AST which might be indication of hepatocyte damage.The finding that significant decrease in total HBV DNA and HBV cccDNA after plasma exchange in ALSS may provide a clue of further research about the fulminant hepatitis B treated with artificial liver support system.