Digital PCR for Absolute Quantification of Adenoviruses

Quantitative real-time PCR assays often lack harmonization, leading to variability in viral load reporting. Differences in calibration materials, assay design, extraction methods, and gene targets further complicate standardization. This study evaluated the Qiagen QIAcuityDx digital PCR (dPCR) platform for absolute quantification of adenoviruses and compared its performance with Bio-Rad droplet dPCR and the standard-of-care quantitative real-time PCR assays. Viral loads of commercially available quality control materials, calibrators, and clinical samples were assessed. Whether adenovirus genotype affects quantification precision across platforms was also examined. Replicates of the human adenovirus (HAdV) verification panel and control materials (Bio-Rad HAdV E4 and ZeptoMetrix HAdV B3) were tested using both dPCR systems. Both demonstrated excellent correlation and equivalent analytical sensitivity at 1000 copies/mL. Remnant clinical samples representing 16 HAdV genotypes were tested in serial dilutions with both digital PCR methods and the standard-of-care assay. Strong agreement was observed across assays for viral loads ≥1000 copies/mL, regardless of genotype. The Qiagen QIAcuityDx platform showed an average bias of -0.31 (SD, 0.44) log copies/mL relative to quantitative real-time PCR. These findings demonstrate that dPCR enables accurate and reproducible HAdV quantification and support its utility for calibration and standardization in clinical molecular testing. Despite minor platform differences, both dPCR systems were highly concordant with each other and with standard-of-care quantification.