Fast and Accurate Multiplex Identification and Quantification of Seven Genetically Modified Soybean Lines Using Six-Color Digital PCR
Alexandra Bogožalec Košir, Sabine Müller, Jana Žel, Mojca Milavec, Allison C. Mallory, David Dobnik
Abstract
The 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.