| Purpose: To develop and establish real-time quantitative PCR method which is specific, sensitive and reliable for detection of HBV cccDNA in liver tissues.Method: The Liver tissues of 37 patients with CHB ,30 patients with liver cancer and serum HBsAg positive, and 7 patients with serum HBV marks and HBV DNA negative were digested using proteinase K to releasing HBV cccDNA and genomic DNA followed by extraction. The extraction products were digested by ATP-Dependent DNase to remove the single strand DNA.HBV cccDNA and tDNA were quantitatively detected by quantitative PCR with selective primers and Taqman probe, respectively. The plasmid pAM6, containing whole HBV genome, were amplified with the primers and fluorescent probe as positive control. Standard curve obtained from serially diluted HBV plasmid pAM6 was used to quantitate HBV cccDNA and tDNA.β-2M, a house-keeping gene was amplified by quantitative PCR to estimate the number of cells as internal control. The liver tissue of serum HBV marks negative was spiked with a known copy number of the plasmid pAM6 followed by extraction and detection of cccDNA and tDNA to evaluate the possible loss rate in the process of extraction.Result: Using our quantitative PCR assay, a linear range of 10~3 to 10~9 copies/assay using primers specific for HBV cccDNA, total HBV DNA (tDNA) andβ-2M was established both in liver tissue and plasmid. In the liver cancer group, the positive rate of cccDNA and tDNA were 73% and 100% respectively. In the CHB group, the positive rate of cccDNA and tDNA were 76% and 89% respectively. In the negative control group,both cccDNA and tDNA were negative. The detection rate ofβ-2M of all samples were 100% .Conclusion: Our fluorescence quantitative real-time PCR assay for HBVcccDNA of liver tissue was accurate, highly specific,highly sensitive and practical. It can be utilized in the quantitative detection of hepatocytes HBV cccDNA as well as in the evaluation of the efficiency of antiviral therapy . |
PDF Full Text Request