Development and in-house validation of two real-time PCR methods for the detection of genome-editing events in soybean FAD2 gene variants

Since the commercialization of the first genetically modified (GM) crops, their relevance in agricultural applications has increased. Soybean (Glycine max) ranks as the most widely cultivated GM crop globally. With the advancements in site-specific genome-editing tools, the development of GM organisms (GMO) using new genomic techniques has accelerated, particularly in agriculture. In the US, a genome-edited soybean variety, Calyno, was developed with altered fatty acid composition by inactivating two members of the fatty acid desaturase 2 (FAD2) gene family, FAD2-1A and FAD2-1B, using TALEN technology. Although deregulated in the US, Calyno soybeans are not authorized for cultivation or import in the European Union and therefore cannot be placed on the European market. To address the need for reliable detection methods in routine analysis for GMO by public authorities, two event-specific qPCR methods were developed to detect the FAD2-1A-Δ63bp and FAD2-1B-Δ23bp gene variants in the Calyno soybeans. In the absence of reference material, methods were validated in-house using synthetic plasmids carrying the target regions’ sequences, following European Network of GMO Laboratories (ENGL) guidelines. Both assays meet the minimum performance requirements for GMO testing, demonstrating high sensitivity, qPCR efficiency, specificity and robustness. An interlaboratory comparison study further confirmed the reliability of these methods. These qPCR assays provide an effective tool for detecting the FAD2-1A-Δ63bp and FAD2-1B-Δ23bp gene variants in Calyno soybeans, supporting GMO testing requirements in the European Union and ensuring accurate monitoring of GM crops.