Development and Validation of a High‐Sensitivity Droplet Digital <scp>PCR</scp> Assay for Serum Hepatitis B Virus <scp>DNA</scp> Detection

Real-time polymerase chain reaction (PCR) is the current standard for serum HBV DNA measurement. However, conventional real-time PCR assays have technical limitations, and are not sensitive enough to detect low-level residual viremia in chronic hepatitis B (CHB) patients. We developed and validated a droplet digital PCR (ddPCR) assay for high-sensitivity detection of serum HBV DNA. A ddPCR assay was developed on the QX200 ddPCR System (Bio-Rad) for detection of serum HBV DNA in 200 μL of serum. The primers and probe were designed to target a highly-conserved region in the HBV X gene. The AcroMetrix HBV Panel (Thermo Fisher Scientific) and CHB patient samples were used for validation experiments to determine the assay sensitivity, specificity, linearity, intra-run variability, and inter-run variability. The ddPCR assay demonstrated lower limit of detection of 1.6 IU/mL and lower limit of quantification of 9.4 IU/mL for serum HBV DNA in probit regression. The assay also achieved excellent specificity (96.2%), linearity (R = 0.994, R²= 0.988, p < 0.001), intra-run variability (mean coefficient of variation [CV]: 0.69%, average intra-run difference: 0.026 log IU/mL), and inter-run variability (mean coefficient of variation [CV]: 4.54%, average inter-run difference: 0.18 log IU/mL). To conclude, we developed a robust ddPCR assay that achieved higher detection sensitivity with lower serum input volume than conventional real-time PCR assays. Our assay may be utilised for measuring residual viremia after nucleos(t)ide analogue therapy or for monitoring patients on novel HBV antivirals.