Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNA from two genetically modified organisms

The application of a ligation-dependent probe amplification (LPA) technique to the simultaneous event-specific detection and relative quantification of DNA from genetically modified organisms in foods is described. The system is based on the ligation of synthetic bipartite probes when hybridized to the corresponding target DNA sequence. The ligation products possess lengths characteristic for each target sequence. Universal primer binding sites (PBS) at the 5 and 3-ends enable their subsequent competitive amplification using one common pair of primers. The use of one fluorescein (FAM) labeled primer permits amplification products to be separated and detected via capillary electrophoresis. Respective probes were designed to allow the detection of reference genes in the genomes from maize and soya, as well as of event-specific junction regions in the transgenic maize line MON810 and in Roundup Ready soya. Specificity, sensitivity, and the potential of the technique for the relative quantification of recombinant DNA were assayed using mixtures of DNA extracted from certified reference maize and soybean flours. The novel strategy results in a modular system which can be complemented by further probes to broaden the range of target sequences.