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

• Published in: Biotechnology letters Vol. 37; no. 10; pp. 2063 - 2073
• Main Authors: Mu, Di, Yan, Liang, Tang, Hui, Liao, Yong
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.10.2015; Springer Nature B.V
• Subjects: Applied Microbiology; Assaying; Biochemistry; Biomedical and Life Sciences; Biotechnology; Cell Line; Cellular; Circularity; Deoxyribonucleic acid; detection limit; Digital; DNA; DNA Primers - genetics; DNA, Circular - analysis; DNA, Circular - genetics; Droplets; Hepatitis; Hepatitis B virus; Hepatitis B virus - genetics; Hepatitis B, Chronic - drug therapy; Hepatitis B, Chronic - virology; Humans; Life Sciences; liver; Liver - virology; Microbiology; monitoring; Original Research Paper; Pathology, Molecular - methods; patients; plasmids; Polymerase Chain Reaction - methods; quantitative polymerase chain reaction; Reproduction; Sensitivity and Specificity; therapeutics; Therapy; Virology - methods; Viruses; Droplet digital PCR; Hepatitis B virus; HepG2.215 cell line; Plasmid-safe ATP-dependent deoxynuclease; Covalently closed circular DNA (cccDNA); cccDNA-specific primer
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-015-1890-5

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. CONCLUSION: 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.