Cross-platform digital PCR evaluation of bovine papilloma virus quantification: introducing PCR-ValiPal for Standardized guided assay validation

Digital Polymerase chain reaction (dPCR) enables precise and absolute quantification of nucleic acids by partitioning samples into thousands of individual PCR micro-reactions. While it minimizes the need for standard curves and enhances reproducibility compared to qPCR, thorough assay validation remains crucial. We introduce PCR-ValiPal, a user-friendly web application that standardizes dPCR assay validation steps and streamlines calculations of limit of blank (LOB), limit of detection (LOD), limit of quantification (LOQ), precision, trueness, and linearity in accordance with International Organization for Standardization (ISO) 20395:2019. To demonstrate PCR-ValiPals capabilities and the value of method-specific optimization, we use it to validate a novel three-color PCR assay for Bovine Papillomavirus (BPV) types 1 and 2, comparing four platforms: Naica (droplet dPCR), QIAcuity (microwell dPCR), LOAA (real-time dPCR), and CFX96 (qPCR). Using synthetic standards, we assess the platforms performance under identical assay conditions. Naica and QIAcuity showed lower LOB and LOQ values, along with minimal bias for BPV-1, while LOAA demonstrated stable but negative bias. Although qPCR exhibited the highest sensitivity for BPV-2, it was less sensitive at low concentrations for BPV-1. These results underscore the value of method-specific optimization and the usefulness of PCR-ValiPal.