In-house validation of a droplet digital PCR system using a multifactorial experimental design

Digital PCR (dPCR, digital polymerase chain reaction) has become an important technology for quantification of nucleic acids and its application in food and feed safety analysis is increasing. International standards such as ISO/IEC 17025 or ISO/IEC 15198 require an appropriate validation of the methods. In this study, a specific approach for the validation and statistical modelling of dPCR systems was developed and applied to the Bio-Rad QX200 Droplet dPCR (ddPCR) system. This approach uses a factorial experimental design and the underlying statistical model reflects the Poisson process governing the measurement mechanism.

We show that most of the experimental factors tested, such as the operator, the primer/probe system and the addition of restriction enzymes, have no relevant effect on the quantification of DNA copy numbers, confirming the robustness of the system. However, the choice of the ddPCR master mix and the droplet volume used to calculate DNA copy concentrations are critical factors. Only with the "Supermix for Probes (no dUTP)" was it possible to confirm the accuracy of the ddPCR system over the entire working range.

High precision, sensitivity, uniformity and robustness of the Bio-Rad QX200 ddPCR system were demonstrated. In addition, we present procedures how to increase the number of stabilised droplets leading to improved acceptance for statistical calculations. The new concept for the in-house validation and modelling of digital PCR systems is transferable to other digital PCR technologies.