Characterisation of the 5′ integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number

Genetically modified (GM) maize event NK603 is in the pipeline for authorisation in the EU and is likely to become a dominating GM maize on the world market. The 5′ integration junction was characterised from a small quantity of commercially available genomic DNA. Specific primers and a probe were designed targeting the 5′ integration junction in the plant, amplifying a 102 bp DNA fragment. A method for specific detection and quantification of NK603 using event-specific real-time PCR based on the identified and cloned sequence is described. The assay was specificity tested against five different GM maizes, one non-GM maize and one GM soya bean. Plasmids were constructed for use as external standards in calibration curves. Due to the lack of certified or other suitable commercial reference material for quantification of NK603, the quantifications were performed on non-target DNA spiked with plasmid DNA containing the cloned NK603 5′ integration junction. The plasmid was detectable down to one copy per PCR. The limits of detection (LOD) and quantitation (LOQ) are estimated to be comparable to those of state-of-the-art methods applied for other GM events.