Development of multiplex real-time PCR assays for screening of genetically modified organisms (GMOs)

The number of commerically available genetically modified organisms(GMOs) and molecular detection techniques have been constantly changing. As aresult, GMO laboratories and the food production industry currently are forced toapply many different methods to reliably test raw material and complex processedfood products. In this study, it was aimed to develop multiplex real-time PCRsystems to detect 35S (the promoter of the cauliflower mosaic virüs), NOS (theterminator of the nopaline synthase gene of Agrobacterium tumefaciens), FMV (the35S promoter from the figwort mosaic virüs), and Bar (from the soilbacteriumStreptomyces hygroscopicus) genes as the most widely used in GMO analysis.Additionally an internal positive control (IPC) indicating the presence or absence ofPCR inhibiting substances, and plant gene specific primer (18sR) for the detection ofplant gene was used in the PCR reactions. Combinations of three 35S, three NOS,one FMV and one Bar TaqManTM probes were tested in Real-time PCR reactions. Asa result, duplex, triplex and quadruflex real-time PCR assays were developedincluding IPC and 18sR genes. All the combinations were performed in both AppliedBiosystem (ABI 7500) and Stratagene (Mx3005P) Real-Time PCR systems. Thedeveloped methods were found to be inexpensive and suitable for fast screening inGMO labs.Keywords: Genetically modified organisms (GMOs), Real-Time PCR, Multiplex