The main objective of the project is to apply and further develop a multiplex DNA detection system for identification of unauthorized genetically modified organisms (GMOs) in European food and feed chains. The relevance of this is underscored by recent incidents with unapproved Bt10 maize, Bt73 rice and LL62 rice, which showed that lab-testing is unavoidable for maintenance of national and EU laws. This requires a reliable, cost-effective detection method. Real time PCR meets sensitivity demands up to a 0.1% level. However, detection and identification of GMOs in food is a time-consuming and expensive puzzle in many cases, when many subsequent real time PCR reactions have to be performed for identification. Padlockprobe, ligation-based multiplex detection does provide a method that can potentially meet all the demands of unauthorized GM detection. It is a three step reaction sequence starting with the detection of all possible targets in a single ligation reaction. Subsequent steps involve amplification by PCR and identification by microarray hybridization. The project has four distinct phases: 1) Identification of the DNA target sequences for padlock probe design. The combination of all available sequence information will be used for the building of a model for unauthorized GMO detection. This model will identify the most informative elements as well as any hiatus in sequence information; 2) Design of padlock probes for the DNA targets. This requires examination of secondary structures compromising the efficiency of the probes, and similarity to known DNA sequences resulting in non-specific reactions. 3) Testing of the padlockprobes in the lab. The most important parameters will be sensitivity, the relation between the sensitivity and the number of targets, and the specificity; 4) Validation of the developed method. Dissemination will occur throughout the project through presentations at (inter)national symposia and papers in peer-reviewed scientific journals.
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