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 HBV cccDNA in PBMC 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.The extractive of HBV DNA positive sera samples from patients with chronic hepatitis B(mild)by were amplified simultaneously to test the specificity of the established fluorescence-based quantitative PCR method.Furthermore, the PBMC 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 PBMC of these patients and were further purified by Plasmid_Safe^TMATP_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 SAS8.1 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)which were purified by Plasmid_SafeTMATP_dependent DNase.HBV cccDNA was detectable in PBMC of the patients with fulminant hepatitis B. The level of HBV cccDNA in PBMC decreased not 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 PBMC of patients with fulminant 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.We find that the level of HBV cccDNA in PBMC after plasma exchange in ALSS decrease without significantly.