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

ABSTRACT 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, R 2 = 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.