title: A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.
abstract: OBJECTIVE
  To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients.
  
  
  RESULTS
  A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples.
  
  
  CONCLUSIONS
  The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.
authors.affiliation: Key Laboratory of Molecular Biology for Infectious Diseases of Ministry of Education, and The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, People's Republic of China. mudi0452@163.com.
authors.forename: Di
authors.initials: D
authors.lastname: Mu
authors.affiliation: Key Laboratory of Molecular Biology for Infectious Diseases of Ministry of Education, and The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, People's Republic of China. yanyan_5212@126.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Yan
authors.affiliation: Key Laboratory of Molecular Biology for Infectious Diseases of Ministry of Education, and The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, People's Republic of China. tangtanghui@gmail.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Tang
authors.affiliation: Institute for Viral Hepatitis, The Second Affiliated Hospital, Chongqing Medical University, Yixueyuan Rd #1, Yuzhong District, P.O. Box #149, Chongqing, 400010, People's Republic of China. yongliao@cqmu.edu.cn.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liao
mesh_terms: D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004270:DNA, Circular / Q000032:analysis* / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology*; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
chemical_list: D017931:DNA Primers; D004270:DNA, Circular
keywords: Covalently closed circular DNA (cccDNA); Droplet digital PCR; HepG2.215 cell line; Hepatitis B virus; Plasmid-safe ATP-dependent deoxynuclease; cccDNA-specific primer