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Development of 10 new screening PCR assays for GMO detection targeting promoters (pFMV, pNOS, pSSuAra, pTA29, pUbi, pRice actin) and terminators (t35S, tE9, tOCS, tg7)

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Abstract

p35S promoter and tNOS terminator are the two primary targets for genetically modified organism (GMO) screening. An increasing number of genetic constructions do not contain p35S and tNOS elements; therefore, new screening assays are required. The use of a larger number of screening methods provides a better coverage of the EU-unapproved GMOs and is a cost-effective approach due to the decrease of tests required for identification. In the present study, new real-time PCR screening assays were developed targeting 10 promoter and terminator elements used in genetically modified constructs: pFMV, pNOS, pSSuAra, pTa29, pUbi, pRice actin, t35S, tE9, tOCS, and tg7. Specificity was verified against different plant species, and the limit of detection was determined on plasmid and genomic reference materials. Criteria of performance were successfully tested taking into account the recommendations of international guidelines. It means that these assays can be considered as ready for an inter-laboratory validation.

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Acknowledgments

This research was conducted within a Belgian research project (Convention RT-06/6 GMODetec) financed by the Belgian Federal Public Service for Public Health, Food Chain Safety, and Environment in an endeavor involving three partners (ISP/WIV, ILVO, and CRA-W). Isabel Taverniers from ILVO (Belgium) kindly provided Arabidopsis DNA, and DNA from A. tumefaciens strains was kindly provided by Enrico Biondi from the University of Bologna (Italy). A part of this study (pUbi and tE9 targets) was financially supported by the UK Food Standard Agency (FSA, contract G03032), and the German Federal Office of Consumer Protection and Food Safety (BVL) through the project GMOseek, under the European ERA-NET consortium SAFEFOODERA. We are grateful to Marjorie Servais and Laurent Timmermans (HELHA Fleurus) for their technical help. We also thank Denis Roulez, Esther Arranz, Nicaise Kayoka-Mukendi, Cécile Ancion, and Gaëlle Antoine from our technical team (GMO team of CRA-W).

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  1. Unit Traceability and Authentication, Department Valorisation of Agricultural Products, Walloon Agricultural Research Center (CRA-W), 24, chaussée de Namur, 5030, Gembloux, Belgium

    Frederic Debode, Eric Janssen & Gilbert Berben

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  1. Frederic Debode
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Correspondence to Frederic Debode.

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Debode, F., Janssen, E. & Berben, G. Development of 10 new screening PCR assays for GMO detection targeting promoters (pFMV, pNOS, pSSuAra, pTA29, pUbi, pRice actin) and terminators (t35S, tE9, tOCS, tg7). Eur Food Res Technol 236, 659–669 (2013). https://doi.org/10.1007/s00217-013-1921-1

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