Triplex PCR Assays for Simultaneous Detection of Maize Ingredients, Maize Allergen-Encoding Genes, and the Cry1Ab Transgene in Processed Foods

Abstract Background Maize is widely used in processed foods but may cause allergic reactions. Reliable detection of maize ingredients, related allergens, and genetically modified organisms (GMOs) in foods is essential for food authenticity, food safety, and consumer protection. However, DNA degradation during food processing and multi-target detection remain significant analytical challenges. Objective To develop and evaluate triplex PCR assays for the simultaneous detection of maize ingredients, allergen-encoding genes, and the Cry1Ab transgene, thereby enabling integrated food authentication, allergen and GMO screening in processed foods. Methods Genomic DNA was extracted from certified reference materials (0–5% MON810 maize) and processed foods using the CTAB method and a commercial plant kit. Two triplex PCR systems were optimized for the detection of species-specific marker (zein), allergen-encoding genes (Zea m 8, Zea m 14), and the Cry1Ab transgene. The PCR products were analyzed via agarose gel electrophoresis. Results The CTAB method yielded higher amounts of amplifiable DNA than the commercial kit. The developed assays enabled simultaneous detection of (i) Zea m 8, Zea m 14, and the zein gene, and (ii) Zea m 8, Zea m 14, and the Cry1Ab transgene. The assays successfully detected 0.1% MON810 reference material in the uniplex and 0.5% in the triplex systems. Analysis of processed foods confirmed reliable detection of all targets despite DNA degradation. Conclusion The proposed triplex PCR assays offer a reliable, rapid, and cost-effective approach for the simultaneous detection of maize ingredients, allergen-encoding genes, and the Cry1Ab transgene, supporting food authentication, allergen and GMO screening in processed foods. Highlights Triplex PCR assays enable rapid, simultaneous detection of maize ingredients, allergen-encoding genes (Zea m 8, Zea m 14, zein) and the Cry1Ab transgene in processed foods, detecting 0.1% and 0.5% MON810 reference materials by uniplex and triplex PCR, respectively.