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Wageningen, Netherlands

Ujhelyi G.,CFRI Central Food Research Institute | van Dijk J.P.,RIKILT Institute of Food Safety WUR | Prins T.W.,RIKILT Institute of Food Safety WUR | Voorhuijzen M.M.,RIKILT Institute of Food Safety WUR | And 5 more authors.
BMC Biotechnology | Year: 2012

Background: With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.Results: Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).Conclusions: From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible. © 2012 Ujhelyi et al; licensee BioMed Central Ltd.

Prins T.W.,RIKILT Institute of Food Safety WUR | van Dijk J.P.,RIKILT Institute of Food Safety WUR | Angeline Van Hoef A.M.,RIKILT Institute of Food Safety WUR | Voorhuijzen M.M.,RIKILT Institute of Food Safety WUR | And 7 more authors.
Food Chemistry | Year: 2010

Current EU regulations on the protection of products with certain characteristics (geographical indications and designations of origin) aim to ensure fair competition for producers and increased consumers' trust. Within the European integrated research project TRACE analytical methods are being developed to allow the maintenance of specific regulations for PGIs (products of protected geographical indication) and PDOs (products of designated origin). An example within the project is the PGI wheat variety Farro della Garfagnana. The aim of the research was to develop a method to establish the purity of Farro della Garfagnana DNA in complex cereal mixtures. The combined approach of padlock probe ligation and multiplex microarray detection can identify possible admixtures. One undesired 'contaminant' for Farro della Garfagnana is common bread wheat (Triticum aestivum), containing the BBAuAuDD genome. Since Farro harbours the BBAuAu genome, absence of the D-genome rules out the presence of bread wheat. The current detection limit of this multimethod is at least 2.5% bread wheat in Farro. © 2008 Elsevier Ltd. All rights reserved.

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