Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Summary

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0-2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5'-sequence and a GMO specific 3'-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5'-nuclease PCR. The presented method is therefore suitable for quantification purposes for food and feed containing the most common maize GMOs.