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LNR NRL NRL - Favv

LNR NRL NRL - Favv

Centre Wallon de

Centre Wallon de Recherches Agronomiques (CRA-W) The Unit Traceability and authentication of CRA-W contributed to the success of the project by its implication in WP1, WP2 and WP4 (development of plasmid reference material). WP1 of the project consisted of the construction of a GMO matrix by collecting all information about the genes, promoters and terminators used in GM constructs. CRA-W supplied the matrix with the information in his possession and participated to the theoretical and practical checking of the information. CRA-W also developed simple Excel-based tools to facilitate the research of information in the matrix or for decision support. This tool will be freely accessible and will be very useful for control or research laboratories. In WP2, the aim was to develop new screening methods. Therefore, CRA-W developed new screening methods based on real-time PCR using TaqMan probes. Two methods targeting the Ubiquitin promoter of maize (pUbi) and the terminator E9 of pea (tE9) were developed and in-house validated taking into account the acceptance criteria such as specificity, sensitivity, limit of detection, efficiency of amplification, rates of false positive and false negative results. The pUbi and tE9 methods were successfully transferred to the LGL laboratory for evaluation. On the other hand, CRA-W evaluated the simplex (p35S-nptII junction) and multiplex methods (duplex PAT/bar, triplex p35S/ tNOS/CTP2-CP4-EPSPS, pentaplex PAT/bar/ p35S/tNOS/CTP2-CP4-EPSPS) methods designed by LGL. Two additional methods were developed at CRA-W and several acceptance criteria were evaluated. In order to avoid false positive results with the tE9 target due to the presence of pea (the tE9 terminator is coming from pea) in a food product, a specific target for pea detection was developed. A last screening target for the Cry1Ab genes was also created and the variations of sequence of this target were studied in the different GM events for which reference material was available. All partners The three NRL-GMO laboratories together with the three other partners were involved in WP4 on the establishment of general guidelines for the validation of qualitative multiplex methods for GMO testing. Based on official IUPAC, ISO and Codex Alimentarius guidelines, a document was drafted as project deliverable, on agreed requirements for method development and in-house validation for a GMO screening method. It was further agreed that these criteria have to be fulfilled before a method goes further to full validation through an official inter-laboratory comparative study. 26

Sylvia.Broeders@wiv-isp.be isabel.taverniers@ilvo.vlaanderen.be Debode@cra.wallonie.be The project participants 27

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