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.
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.
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Acknowledgments
The work of current report was supported in part by the National Natural Science Foundation of China (NSFC 81172066; NSFC 81472858) and the start-up fund from the Second Affiliated Hospital, Chongqing Medical University (to Y. Liao). In addition, we would like to express our sincere thanks to Dr. Bin Peng, Center for Research of Medicine and Social Development, Chongqing Medical University, for his kind help on statistical data analysis. We also thank Dr. Walter N Hittelman (at The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA) for language improvement of the manuscript.
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Mu, D., Yan, L., Tang, H. et al. 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. Biotechnol Lett 37, 2063–2073 (2015). https://doi.org/10.1007/s10529-015-1890-5
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DOI: https://doi.org/10.1007/s10529-015-1890-5