Fast and accurate multiplex identification and quantification of seven genetically modified soybean lines using six-color digital PCRBogožalec Košir, Alexandra (Avtor) Muller, Sabine (Avtor) Žel, Jana (Avtor) Milavec, Mojca (Avtor) Mallory, Allison C. (Avtor) Dobnik, David (Avtor) digital PCRdPCRquantificationmultiplexinggenetically modified organisms6-color systemvirus diagnosticsvirologyThe proliferation of genetically modified organisms (GMOs) presents challenges to GMO testing laboratories and policymakers. Traditional methods, like quantitative real-time PCR (qPCR), face limitations in quantifying the increasing number of GMOs in a single sample. Digital PCR (dPCR), specifically multiplexing, offers a solution by enabling simultaneous quantification of multiple GMO targets. This study explores the use of the Naica six-color Crystal dPCR platform for quantifying five GM soybean lines within a single six-plex assay. Two four-color assays were also developed for added flexibility. These assays demonstrated high specificity, sensitivity (limit of detection or LOD < 25 copies per reaction) and precision (bias to an estimated copy number concentration <15%). Additionally, two approaches for the optimization of data analysis were implemented. By applying a limit-of-blank (LOB) correction, the limit of quantification (LOQ) and LOD could be more precisely determined. Pooling of reactions additionally lowered the LOD, with a two- to eight-fold increase in sensitivity. Real-life samples from routine testing were used to confirm the assays’ applicability for quantifying GM soybean lines in complex samples. This study showcases the potential of the six-color Crystal dPCR platform to revolutionize GMO testing, facilitating comprehensive analysis of GMOs in complex samples.20232024-03-29 11:42:31Neznano18583UDK: 578ISSN pri članku: 2304-8158DOI: 10.3390/foods12224156COBISS_ID: 173360131sl