ABSTRACT
Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.