Pei-Xue Jiang1, Ri-Cheng Mao1, Min-Hui Dong1, Xue-Ping Yu1, Qi Xun1, Jin-Yu Wang1, Li Jing1, Deng Qiang2, Ji-Ming Zhang3. 1. Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China. 2. Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China. 3. Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China. Electronic address: jmzhang@fudan.edu.cn.
Abstract
BACKGROUND: Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA. METHODS: Exonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies. RESULTS: A linear range of 101-107 copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001). CONCLUSIONS: The real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice.
BACKGROUND:Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA. METHODS: Exonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies. RESULTS: A linear range of 101-107 copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001). CONCLUSIONS: The real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice.