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LNR NRL NRL - Favv

LNR NRL NRL - Favv

NRL-GMO: GMOSeek research project (2009 – 2011) for GMO detection IPH: Sylvia Broeders, Elodie Barbau-Piednoir, Guillaume Mbongolo Mbella, Nina Papazova, Antoine Pouppez, Nancy Roosens ILVO: Isabel Taverniers, Marc De Loose CRA-W: Frédéric Debode, Gilbert Berben, Eric Janssen Introduction The GMOseek project (SAFEFOODERA: “Food Safety – forming a European platform for protecting consumers against health risks”) was financed by the European Commission under the ERA-NET platform for protecting consumers against health risks and was running from 1/06/2009 till 31/05/2011. It consisted of six European partners from four different member states: Centre Wallon de Recherches Agronomiques (CRA-W, BE), Instituut voor Landbouw- en Visserijonderzoek (ILVO, BE), Wetenschappelijk Instituut Volksgezondheid (WIV-ISP, BE), Bayerische Landesamt für Gesundheit und Lebensmittelsicherheit (LGL, DE), Joint Research Centre – Institute for Health and Consumer Protection (JRC-IHCP, IT) and Nacionalni Inštitut za Biologijo (NIB, SI). The three Belgian laboratories which took part in the project compose further the National Reference Laboratory for Genetically Modified Organisms (NRL-GMO). The project consisted of different work packages (WP) and each laboratory participated in at least 2 work packages. The project dealt with the detection of Genetically Modified Organisms (GMO). Given the extensive legislation concerning GMO’s, the fast increasing amount of authorized GMO’s being placed on the market and the growing chance of the presence of unauthorized GMO’s (UGM), it is necessary to develop new detection techniques and to refine the existing ones. The principal results of the project were: • Construction of a database gathering all information on existing GM events • Establishment of a bio-informatics system that allows selecting a maximal number of EU-authorized GM events based on the screening of an optimal set of genetic elements • Development and in-house validation of SYBRGreen qPCR screening methods • Development and in-house validation of TaqMan qPCR screening methods • Development and in-house validation of a pentaplex qPCR screening method • Transfer of the new qPCR screening methods to a partner laboratory • Establishment of a document concerning validation guidelines for qualitative qPCR methods Hereafter, a small outline of the work done by the three Belgian NRL-GMO laboratories is given. 24

Wetenschappelijk Instituut Volksgezondheid (WIV-ISP) The GMOlab of the unit Platform Biotechnology and molecular Biology of the WIV-ISP was involved in the GMOseek project in work packages WP2 and WP4. In WP2, the work of the lab consisted in the extraction and characterization of genomic DNA (gDNA). Hereto Certified Reference Materials (CRM) were purchased and gDNA was isolated using extraction methods that are being applied on European level. The lab could show that the DNA extracted from the different materials was sufficient to cover the needs of the partner laboratories to develop their methods, that it was of good quality and that no inhibition was present. The extracted gDNA material was distributed to the partners according to their needs. Additionally, a plasmid was constructed for each method developed and validated by the GMOlab. In WP4, the GMOlab developed and validated three new screening methods targeting the genetic elements pNOS, pFMV and Cry3Bb which are present in different GM events. These methods allow covering more GM events and to increase the discriminating power of the previously validated and patented ‘Combinatory SY- BRGreen qPCR screening’ (CoSYPS) system. The system allows determining which GM events could possibly be present in a sample through a combination of genetic elements and is used for routine analysis of food and feed samples in the GMOlab under ISO 17025 accreditation. For each new genetic marker, the GMO lab could show that the parameters, that need to be evaluated during in-house validation, were within the acceptance criteria. All information was gathered in a validation dossier. In a last step, the three methods were transferred to the partner laboratory JRC-IHCP together with the necessary materials and the protocol to follow. Comparison of the results from both laboratories showed the reproducibility of the three methods which is important seen the harmonization of detection methods the EU is aiming for. Finally, it could be concluded that the three new screening methods were fit for the detection of GMO in food and feed samples and could be integrated in the CoSYPS system allowing a more cost and time efficient GMO detection. Instituut voor Landbouw- en Visserijonderzoek (ILVO) ILVO contributed in three of the GMOseek project work packages: WP1 on Bioinformatics, WP3 on DNA-based hybridization methods, and WP4 on Reference material and Guidelines for validations. Within WP1, an in-house “GMOmatrix” database was set up containing relevant information of all GM events existing worldwide. Regular check-up and maintenance of this database formed one of the tasks where ILVO was actively involved in. Each six months, all GM events (records) in the database were checked for completeness and correctness, errors were indicated and novel data on GM events or novel GM events were added into the database. This database further formed the basis for the development of novel bioinformatics tools, useful in the selection and design of GMO screening methodologies as a first step in official laboratories’ GMO testing procedures. The task within WP3 entitled ‘NAIMA platforms for multiplex screening on microarray’, aimed at optimising and evaluating the NASBA-Integrated Multiplex Amplification (NAIMA) screening technology developed by NIB (Ljubljana, Slovenia). After optimisation and in-house validation, done at NIB, ILVO acted as transfer lab for further testing the hexaplex method. ILVO performed amplification reactions (NASBA) and hybridization of the reactions to pre-designed microarray plates, using materials and following the exact protocols from NIB. However the arrays were sent back to NIB for scanning and data analysis. Based on the low robustness and difficult transfer and implementation of the technology in general, the NAIMA multiplex platform was not considered as relevant for further inter-laboratory testing. 25

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