Evaluation of Systems for Nopaline Synthase Terminator in Fast and Standard Real-Time PCR to Screen Genetically Modified Organisms

Abstract

The increasing number and diversity of genetically modified organisms (GMOs) developed and commercialised forces laboratories to apply many different methods of analysis. Matrix-based approach helps to minimize analytical effort reducing the number of identifications. However, the correctness of screening phase needs efficient methods. In this paper, 15 systems for the nopaline synthase terminator (T-nos) from Agrobacterium tumefaciens, a genetic element present in several genetically modified (GM) plants, were tested. The systems were obtained from three methods, and their primers and probes were combined and tested in real-time polymerase chain reaction (PCR) in fast and standard mode. The results have showed that the fast mode presented the lowest mean quantification cycle (Cq) and the highest ∆Rn, that is, the difference between normalized reporter and baseline. These parameters of system efficiency prove that the fast real-time PCR is a possible approach to obtain good data in less than half the time compared to standard mode. The proposed approach allows a practical way to evaluate the most efficient set of oligonucleotides (e.g., primers and probe) in fast and standard real-time PCR before validation. This article describes also in-house validation of the best set oligonucleotides.