Validation of a Duplex Digital PCR Assay for the Quantification of the NK603 Maize Event Across Three dPCR Platforms.

In the European Union, mandatory labeling of food and feed products is required when authorized genetically modified organisms (GMOs) exceed 0.9% per ingredient, necessitating reliable analytical methods for official control laboratories. Event-specific PCR assays validated according to ISO/IEC 17025 are the reference approach for GMO detection, identification, and quantification. The growing use of digital PCR (dPCR) has encouraged the adaptation of real-time PCR methods to dPCR-based strategies, as dPCR enables absolute quantification without calibration standards, shows reduced sensitivity to inhibitors, and allows for the design of a multiplex assay. In this study, an in-house validation of a duplex dPCR assay targeting the maize GM event NK603 and the HMG reference gene was performed on three platforms: Bio-Rad QX200™ (Pleasanton, CA, USA), Qiagen QIAcuity (Venlo, The Netherlands), and Thermo Fisher QuantStudio Absolute Q (Waltham, MA, USA). All validation parameters met the Joint Research Centre (JRC) acceptance criteria. In particular, this assay demonstrated high specificity, sensitivity (limit of quantification or LOQ < 35 copies per reaction), precision, and trueness (RSDr and bias <25%). The data indicate that the duplex dPCR assay can be used for routine GMO analysis and future collaborative validation studies.