Quantitation Of Hepatitis B Virus Covalently Closed Circular DNA (cccDNA) In Chronic Hepatitis B Patients

Chronic hepatitis B virus (HBV) infection remains a major health problem affecting approximately 400 million people worldwide. Formation of HBV cccDNA begins from the entry of HBV gemonic DNA (relaxed circular DNA) into the nucleus and is believed to the reason for HBV persistent infection. HBV cccDNA is reported to be resistant to conventional antivirus patients including lamivudine and interferon-α which can significantly decrease serum HBV DNA level, and plays a crucial role of the relapse after the withdrawal of antivirus therapy.Because of the importance of HBV cccDNA in the mechanisms of chronic Hepatitis B and its therapy, establishment of quantitation method has been a focus for many years and is developed to the real-time PCR assay at present. However there are still many problems in the detection method, among those is the false positiveness caused by the amplification of other replicative intermediates including rcDNA and ssDNA, Besides, the sensitiveness ,accuracy and simplicity of the method are also expected to be improved.Our quantitative method contains such major protocols: Liver tissue previously cut and weighed to about 10mg was digested with proteinase K in a lysis buffer. DNA was extracted by phenol/chloroform-based method and then...