Development of a modular system for detection of genetically modified organisms in food based on ligation-dependent probe amplification

Abstract

The application of a method based on ligation-dependent probe amplification to the simultaneous detection of different genetically modified organisms in food is described. The ligation reaction of target-specific probes with characteristic lengths and universal primer binding sites is followed by PCR amplification using fluorescein-labeled primers. The separation and the detection of DNA fragments are achieved by capillary electrophoresis via laser-induced fluorescence. The approach allows the simultaneous detection of several targets corresponding to different levels of specificity in a one-tube assay. Synthetic oligonucleotides were designed to detect (1) reference genes in the genome from maize, soya and rapeseed, (2) the CaMV 35S-promotor as screening element, (3) the construct-specific 35S-pat junction, and (4) the event-specific regions of the transgenic maize line MON 810 and of Roundup Ready soya. Specificity and sensitivity (GMO content 0.1%) of the approach were shown for all targets. This novel analytical strategy represents a flexible, modular system for the surveillance of GMO-derived products that can be readily complemented by further target sequences of interest.