title: An innovative and integrated approach based on DNA walking to identify unauthorised GMOs.
abstract: In the coming years, the frequency of unauthorised genetically modified organisms (GMOs) being present in the European food and feed chain will increase significantly. Therefore, we have developed a strategy to identify unauthorised GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. This integrated approach is performed in two successive steps on Bt rice grains. First, the potential presence of unauthorised GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S pCAMBIA element. Second, its presence is confirmed via the characterisation of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. This approach allows to rapidly identify the transgene flanking region and can easily be implemented by the enforcement laboratories.
authors.affiliation: Scientific Institute of Public Health (WIV-ISP), Platform of Biotechnology and Molecular Biology (PBB), J. Wytsmanstraat 14, 1050 Brussels, Belgium; Scientific Institute of Public Health (WIV-ISP), Biosafety and Biotechnology Unit (SBB), J. Wytsmanstraat 14, 1050 Brussels, Belgium; Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Sciences Unit, Burg. Van Gansberghelaan 115, bus 1, 9820 Merelbeke, Belgium; University of Gent (UGent), Faculty of Pharmaceutical Sciences, Laboratory of Pharmaceutical Biotechnology, Harelbekestraat 72, 9000 Ghent, Belgium.
authors.forename: Marie-Alice
authors.initials: MA
authors.lastname: Fraiture
authors.forename: Philippe
authors.initials: P
authors.lastname: Herman
authors.forename: Isabel
authors.initials: I
authors.lastname: Taverniers
authors.forename: Marc
authors.initials: M
authors.lastname: De Loose
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.forename: Nancy H
authors.initials: NH
authors.lastname: Roosens
mesh_terms: D016386:Chromosome Walking / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D030841:Food, Genetically Modified / Q000592:standards*; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: DNA walking; GM Rice; GMO detection; Integrated approach; Quantitative real-time PCR; Transgene flanking region; Unauthorised GMO; pCAMBIA vector