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