Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening too

A GG Nucleotide Sequence of the 3′ Untranslated Region of Amyloid Precursor Protein mRNA Plays a Key Role in the Regulation of Translation and the Binding of Proteins

The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3′ untranslated region (3′UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3′UTR containing 8 nucleotides more than the short 3′UTR allows the recovery of an efficiency of translation similar to that of the long 3′UTR. Moreover, the two guanine residues located at the 3′ ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3′UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3′UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation

NRL-GMO: GMOSeek research project (2009 - 2011) for GMO detection361

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