| This research was composed of three parts: I . Establishment of a new assay for quantitative detection of intracellular hepatitis B virus cccDNA; Effect of oxymatrine on intracellular hepatitis B virus cccDNA of HepG2.2.15 cell line; Detection of cccDNA in peripheral blood mononuclear cells of patients chronically infected with hepatitis B virus.1. Establishment of a new assay for quantitative detection of intracellularhepatitis B virus cccDNAObjective To establish a new assay for quantitative detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA ) in infected hepatocytes. Methods The Hepatitis B virus transfected hepatoblastoma cell line HepG2.2.15 which can consistently produce Dane particles was maintained in DMEM medium containing 380g/ml G418 and 10% fetal bovine serum .Cells in the exponential period were harvested from flasks, then intracellular HBV cccDNA was extracted from pellet containing 1 106 cells with mini plasmid extraction kit. The extraction product was further purified by mung bean nuclease to remove the residual HBV relaxed circular DNA. HBV cccDNA was quantitatively detected by fluorescent PCR with selective primer set and Taqman MGB probe. Culture medium before exponential period, HBV DNA positive and negative serum samples from patients with chronic hepatitis B (mild) were amplified simultaneously to test the specificity of the fluorescent PCR method. Plasmids with target HBV fragment inserted were amplified with the same primer set and fluorescent probe to determine the sensitivity of the method. Results HBV cccDNA was detectable in HepG2.2.15, and the average quantity was 18 copies per cell approximately. No detectable fluorescent signal was observed when culture medium and serum samples were amplified. The detectable HBV cccDNA was as low as 103 copies per millilitre at least by this method. Conclusions This method can be utilized in quantitative detection of intracellular HBV cccDNA. It is convenient, highly specific and highly sensitive.
|
PDF Full Text Request