Exonuclease Ⅰ and Ⅲ improve the detection efficacy of hepatitis B virus covalently closed circular DNA

作     者：Pei-Xue Jiang Ri-Cheng Mao Min-Hui Dong Xue-Ping Yu Qi Xun Jin-Yu Wang Li Jing Deng Qiang Ji-Ming Zhang 

作者机构：Department of Infectious DiseasesHuashan HospitalFudan UniversityShanghaiChina Department of Pathobiology and Key Laboratory of Medical Molecular VirologySchool of Basic Medical SciencesFudan UniversityShanghaiChina 

出 版 物：《Hepatobiliary & Pancreatic Diseases International》 (国际肝胆胰疾病杂志（英文版）)

年 卷 期：2019年第18卷第5期

页      面：458-463页

核心收录：

学科分类：1004[医学-公共卫生与预防医学(可授医学、理学学位)] 100401[医学-流行病与卫生统计学] 10[医学] 

基　　金：supported by grants from National Natural Science Foundation of China(81471933 and 81672009) the National Science and Technology Major Project for Infectious Diseases(2013ZX10002-001) Shanghai Innovation and Development Fund(15DZ1940803) 

主　　题：Hepatitis B virus cccDNA Selective Sensitive 

摘      要：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 10 1 –10 7 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.