Quantification of intrahepatic cccDNA in HBV associated hepatocellular carcinoma by improved ddPCR method

The quantification of intrahepatic covalently closed circular DNA (cccDNA) is important for assessing the efficiency of anti-HBV therapy. Exonuclease treatment is essential before real-time quantitative PCR (qPCR) or droplet digital PCR (ddPCR) measurement to improve the specificity of cccDNA quantification. In this research, we compared the limit of detection (LOD) of qPCR and ddPCR and evaluated the digestion efficiency of three exonuclease treatments, PSAD, exonuclease III and T5 exonuclease, when measuring cccDNA in cells or clinical samples by ddPCR. We demonstrated that the LOD of ddCPR was 5.9 copies/reaction, which was much lower than that of qPCR (54.9 copies/reaction), indicating that ddPCR is more sensitive than qPCR. Meanwhile, compared to PSAD or Exo III, UNG and T5 exonuclease treatment combined with ddPCR is more effective in detecting intrahepatic cccDNA in clinical samples. Finally, the median intrahepatic cccDNA was 2.6 copies/10⁴ cells in 26 pairs of HCC samples determined by the improved ddPCR method. Therefore, we developed an optimized ddPCR method, which can be used for the absolute quantification of low levels of intrahepatic cccDNA more precisely.