Establishment Of Rolling Circle Amplification Method For Detection Of Hepatitis B Virus Cccdna

Objective To detect HBV covalently closed circular DNA by rolling circle amplification (RCA)method, and study the specificity and sensitivity of this method.Methods Wild-type full-length HBV genomes plasmid was digested with restriction enzyme,then treated with T4 DNA ligase and concentrated. The DNA fragment as standard HBV cccDNA was recovered by the nucleic acid purification kit (Axygen). Total DNA was extracted from infected hepatic tissue with QIAamp DNA Mini Kit (QIAGEN). The extraction product was further purified by Plasmid SafeTM ATP dependent Dnase(Epicentre). RCA method was used to amplify genomes from samples, including standard HBV cccDNA, 3.2Kb liner HBV DNA, infected hepatic tissue samples, HBV negative hepatic tissue and 15 HBV positive serum samples. We used two steps of agarose gel electrophoresis and Southern Blot to validate the product. Ten-fold serial dilutions of standard HBV cccDNA were amplified to determine the sensitivity of the method.Results The standard HBV cccDNA was constructed successfully and could be detectable by RCA method.HBV cccDNA could be detected from HBV positive hepatic tissue of at least 2mg. No detectable product was observed in 3.2Kb liner HBV DNA, HBV negative hepatic tissue and HBV positive serum samples.Conclusions RCA method proved to be effective for rapid and sensitive detection of HBV cccDNA with high specificity and sensitivity.