Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices.
BACKGROUND
In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element.


METHODS
Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system.


RESULTS
First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples.


CONCLUSIONS
Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.
Scientific Institute of Public Health (WIV-ISP), Platform of Biotechnology and Molecular Biology (PBB) and Biosafety and Biotechnology Unit (SBB), J. Wytsmanstraat 14, 1050, Brussels, Belgium. MarieAlice.Fraiture@wiv-isp.be.|Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Sciences Unit, Burg. Van Gansberghelaan 115 bus 1, 9820, Merelbeke, Belgium. MarieAlice.Fraiture@wiv-isp.be.|Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Pharmaceutical Biotechnology, Ottergemsesteenweg 460, 9000, Ghent, Belgium. MarieAlice.Fraiture@wiv-isp.be.
Marie-Alice
MA
Fraiture
Scientific Institute of Public Health (WIV-ISP), Platform of Biotechnology and Molecular Biology (PBB) and Biosafety and Biotechnology Unit (SBB), J. Wytsmanstraat 14, 1050, Brussels, Belgium. philippe.herman@wiv-isp.be.
Philippe
P
Herman
Scientific Institute of Public Health (WIV-ISP), Platform of Biotechnology and Molecular Biology (PBB) and Biosafety and Biotechnology Unit (SBB), J. Wytsmanstraat 14, 1050, Brussels, Belgium. Loic.Lefevre@wiv-isp.be.
Loic
L
Lefèvre
Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Sciences Unit, Burg. Van Gansberghelaan 115 bus 1, 9820, Merelbeke, Belgium. Isabel.Taverniers@ilvo.vlaanderen.be.
Isabel
I
Taverniers
Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Sciences Unit, Burg. Van Gansberghelaan 115 bus 1, 9820, Merelbeke, Belgium. marc.deloose@ilvo.vlaanderen.be.|Ghent University, Faculty of Sciences, Department of Plant Biotechnology and Bioinformatics, Technologiepark 927, 9052, Ghent, Belgium. marc.deloose@ilvo.vlaanderen.be.
Marc
M
De Loose
Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Pharmaceutical Biotechnology, Ottergemsesteenweg 460, 9000, Ghent, Belgium. Dieter.Deforce@UGent.be.
Dieter
D
Deforce
Scientific Institute of Public Health (WIV-ISP), Platform of Biotechnology and Molecular Biology (PBB) and Biosafety and Biotechnology Unit (SBB), J. Wytsmanstraat 14, 1050, Brussels, Belgium. nancy.roosens@wiv-isp.be.
Nancy H
NH
Roosens
D016386:Chromosome Walking / Q000379:methods*; D018744:DNA, Plant / Q000235:genetics*; D030841:Food, Genetically Modified; D006801:Humans; D030821:Plants, Genetically Modified; D060888:Real-Time Polymerase Chain Reaction
D018744:DNA, Plant