Standardized digital PCR assay validation using PCR-ValiPal, demonstrated in cross-platform quantification of bovine papilloma virus

Abstract

Background: Digital PCR (dPCR) enables precise and absolute quantification of nucleic acids by partitioning samples into thousands of reactions, improving reproducibility and reducing reliance on standard curves compared to qPCR. However, rigorous assay validation remains essential to ensure reliability, particularly for parameters such as limit of detection, limit of quantification, trueness, and linearity. Existing guidelines (e.g.,MIQE, dMIQE, ISO 20395:2019) highlight these requirements, but implementation is laborious and inconsistent across laboratories. To address this, we developed PCR-ValiPal, a user-friendly web application that standardizes and streamlines dPCR assay validation and reporting. Results: PCR-ValiPal calculates the full range of analytical parameters required for ISO-compliant assay validation, including limit of blank, limit of detection, limit of quantification, precision, trueness, and linearity. While broadly applicable to any nucleic acid target, we demonstrate its use with a three-color PCR assay for bovine papillomavirus (BPV), a clinically relevant representative DNA assay, types 1 and 2, benchmarked across four platforms: Naica (droplet dPCR), QIAcuity (microwell dPCR), LOAA (real-time dPCR), and CFX96 (qPCR). Cross-platform comparisons revealed Naica and QIAcuity achieved low LOB and LOQ values with minimal bias, while LOAA exhibited stable but negative bias. qPCR performed best for BPV-2 sensitivity but was less reliable for BPV-1 at low concentrations. These results illustrate both the value of platform-specific optimization and the utility of PCR-ValiPal in providing transparent, standardized validation outputs. Significance: PCR-ValiPal supports transparent, reproducible, and ISO-aligned validation of PCR-based assays, lowering barriers for both expert and non-expert users. By centralizing statistical analyses in a single tool, it enables reliable comparison across platforms and targets, facilitating adoption in research, diagnostics, and regulatory contexts. This work underscores the importance of standardized validation for ensuring confidence in nucleic acid quantification.