Critical Factors Affecting Digital PCR Quantification Accuracy in HBV DNA Reference Material for In Vitro Diagnostics.

Digital PCR, as an advanced technique for absolute nucleic acid quantification, has been widely used in the value assignment and characterization of reference materials, including in vitro diagnostic reference standards. However, few studies have systematically evaluated the impact of different digital PCR platforms and nucleic acid extraction kits on the quantification of reference materials. In this study, we established a highly specific and reproducible digital PCR assay for the hepatitis B virus and systematically evaluated the influences of commercial digital PCR platforms, primer-probe sets, and nucleic acid extraction kits on reference materials quantification. Results demonstrated good consistency in quantitative measurements across mainstream commercial digital PCR platforms based on different technical principles, with a mean interplatform coefficient of variation (CV) of 9.05%. Notably, nucleic acid extraction efficiency demonstrated the greatest impact on quantification accuracy, with a mean CV across different extraction kits of 76.66%. Primer-probe design also contributed substantially to the measurement uncertainty. These findings provide important insights that support the standardization of digital PCR protocols for reference materials quantification.