A Duplex Digital PCR Method for Quantification of Transgene Soybean Line MON40-3-2 in Complex Matrices

The widespread use of genetically modified organisms (GMOs) since 1994 has led to increased regulatory scrutiny to monitor their presence in supply chains. For many countries, strict adherence to regulatory thresholds for labeling GMO-containing products is crucial. However, detecting and quantifying GMOs in complex food and feed matrices poses challenges. While real-time quantitative PCR (qPCR) has been the gold standard for GMO detection, it can face limitations, especially in the presence of inhibitors. Recognizing these challenges, digital PCR (dPCR) emerges as a promising alternative, offering absolute quantification without the need for standard curves and mitigating the impact of inhibitors. This chapter outlines a duplex dPCR method targeting a specific genetically modified soybean line MON40-3-2 and soybean reference gene (Le1), providing several practical advantages over simplex dPCR. By allowing simultaneous amplification of two target sequences, the duplex method reduces time, reagents, and resources, while ensuring consistency and reliability of results. Although described for the QX200 platform, the method is adaptable to other platforms with caution.