Heat processing compromises GMO detection in soybean-enriched biscuits.

The widespread adoption of genetically modified (GM) crops necessitates robust detection methods, particularly for processed foods, where DNA degradation compromises analytical reliability. This study investigated the effect of industrial baking temperatures (190–210 °C) on the detectability of Roundup Ready® soybean (GTS 40-3-2) DNA in a biscuit matrix containing 0–100% GM soybean flour. Real-time PCR analyses targeted the soybean-specific lectin gene, the CaMV 35 S promoter, and the cp4 epsps transgene. The results demonstrate that thermal processing induces sequence-specific DNA degradation, which is more pronounced for the 35 S promoter and cp4 epsps sequences than for the lectin reference gene. This observation indicates that the assumption of equivalent amplification behavior between target and reference sequences—a fundamental premise of comparative qPCR approaches such as the ΔΔCq method—may not consistently hold in thermally processed matrices. Near the European Union’s 0.9% labeling threshold, such differential degradation patterns may increase the risk of false-negative results. Overall, this study demonstrates that the principal challenge in the analysis of processed foods lies not in absolute GMO quantification per se, but in the reliable interpretation of detection results under conditions of sequence-specific DNA degradation. These findings underscore the need for matrix-optimized analytical protocols tailored to processed foods and for a critical re-evaluation of current qPCR-based quantification frameworks in this context.