The Establishment Of HBV CccDNA Quantitation Methods And Clinical Application

This research was composed of two parts:â… .Quantitation of HBVcccDNA in serum and liver tissues;â…¡.Characteristic of HBV repilication in CHB,LC and HCC patients.â… .Quantitation of hepatitis B virus cccDNA in serum and liver tissuesAIM:To establish a method for quantitative detection of HBV cccDNA with high sensitivity and specificity.METHODS:Design two pairs of primers and probes for HBV total DNA and HBV cccDNA.The primers for HBV cccDNA flank the gap region and the incomplete strand of DNA.Plasmid standard gradient dilution tested and verified the sensitivity and stability of this method,and established the linear range of detection about two pairs of primers and probes.According to different digestion method the samples was divided into no enzyme group,restriction endonuclease digestion group,Plasmid-Safe-ATP-Dependent DNase digestion group,restriction endonuclease with Plasmid-Safe-ATP-Dependent DNase digestion group.Compared the result of different groups in order to evaluate the specificityof the method.RESULTS:Improved the current detection method,the lowest standard sample accurately and repeatedly quantified contained 10² copies of HBV DNA/mL in HBV cccDNA and HBV total DNA detection.Intra-assay reproducibility was assessed by repeating plasmid standard gradient dilution quantitation.The samples were tested three times in a single run of HBV total DNA PCR and HBV cccDNA PCR respectively,and gave coefficients of variation(CV) both below 3%.Inter-assay variability was also assessed by testing plasmid standard gradient dilution samples once in each of seven different runs of HBV total DNA PCR and HBV cccDNA PCR respectively.The coefficients of variation obtained for HBV total DNA PCR below 4%,and for HBV cccDNA PCR below 3%.The amplification efficiency of 13F,71F and commercial kit of quantitation HBV total DNA were same.By contrast,the serum sample was digested with Plasmid-Safe-ATP-Dependent DNase firstly,then was amplified by 71F which was better than other methods and could reduce 4log₁₀copies/ml non-specific amplification;the liver biopsy sample was digested with restriction endonuclease MluI and Plasmid-Safe-ATP-Dependent DNase firstly,then was amplified by 71F which could reduce 5log₁₀copies/ml non-specific amplification.CONCLUSIONS:Apply a new method to process samples which can improve the specificity of HBV cccDNA quantitation.Establish a method for HBV cccDNA quantitation with high sensitivity and specificity. â…¡.Characteristic of HBV repilication in CHB,LC and HCC patientsAIM:To quantify hepatitis B virus(HBV) total DNA and covalently closed circular DNA(cccDNA) in liver biopsies and sera which from Chronic hepatitis B(CHB),liver cirrhosis of hepatitis B(LC) and hepatitis B relevance hepatocellular carcinoma(HCC) patients,and analyze HBV replication under the different circumstances of diseases.METHODS:Total HBV DNA and HBV cccDNA in serum and liver biopsy samples were measured in 21 CHB,23 LC and 25 HCC patients by the real-time PCR assay.RESULTS:The levels of total HBV DNA in serum,intrahepatic total HBV DNA,intrahepatic HBV cccDNA,as well as the proportion of intrahepatic HBV cccDNA in total HBV DNA decreased progressively in CHB,LC and HCC,moreover CHB had significantly higher levels of total HBV DNA in serum and liver biopsy samples than HCC(log[total serum HBV DNA]P=0.013;log[total intrahepatic HBV DNA]P=0.014);CHB and LC had significantly higher levels of intrahepatic HBV cccDNA and the proportion of intrahepatic HBV cccDNA in total HBV DNA than HCC(P＜0.01);HBV cccDNA couldn't be detected in serum of all patient.In CHB,the levels of serum's total HBV DNA,intrahepatic total HBV DNA and HBV cccDNA in HBeAg-positive group had significantly higher than the HBeAg-negative group(P＜0.01),but in LC only intrahepatic total HBV DNA and HBV cccDNA had statistical difference between HBeAg-positive and negative group(P=0.014),no statistical difference between HBeAg-positive and negative group in HCC.The relevance of total HBV DNA in serum,intrahepatic total HBV DNA and intrahepatic HBV cccDNA was decreased gradually in CHB,LC and HCC.Whether CHB,LC or HCC,the serum total HBV DNA,intrahepatic total HBV DNA or intrahepatic HBV cccDNA levels were found to have no correlation with ALT and TBIL levels.CONCLUSIONS:When CHB progressed to LC and HCC,HBV replication activity decreased.Duplication of HBV in LC was lower than CHB but had no statistical difference.The levels of HBV reproduce in HBeAg-positive group was higher than HBeAg-negative group in CHB,LC and HCC.