Improved methods for detecting genetically modified components in puffed cereal products: Discovering genetic insights via PCR-RFLP, Real-time PCR, and Sanger sequencing

Real-time polymerase chain reaction (PCR) assays require specialized equipment that may not be available in some laboratories; this study employed a sensitive gel-based PCR method. The PCR-restriction fragment length polymorphism (RFLP) technique has gained popularity due to its accuracy in identifying transgenic varieties. Sanger sequencing was utilized to identify genetic elements, while TaqMan real-time PCR was employed to confirm specific constructs in GMOs. Iran has rules in place to ensure the safety of GMOs. However, many consumers do not know much about these products, and there is not enough clear information about their identification and biological characteristics. This study fills in missing information by using molecular methods to identify genetically modified (GM) parts in puffed cereal products. It identifies important markers like the cauliflower mosaic virus 35S promoter (CaMV P35S), neomycin phosphotransferase II (NPT II), MON 810, and GTS 40-3-2. We analyzed 384 samples using a method called PCR-RFLP to study CaMV P35S and NPT II. Furthermore, 50 samples were tested using TaqMan real-time PCR to verify the presence of GTS-40-3-2. Among 384 samples from Isfahan, 24.3% contained CaMV P35S, 27% had NPT II, and 38.8% were positive for MON810. Furthermore, 20% of the 50 samples contained GTS-40-3-2. • A suitable DNA isolation method was introduced for samples exposed to intense industrial processing. • A sensitive gel-based PCR method was optimized. • TA cloning of PCR products was refined for sequencing applications. • Silver nitrate staining was optimized for RFLP confirmation. • Sanger sequencing was utilized to validate PCR results. • TaqMan Real-Time PCR was performed for event identification.