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Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure
Authors:Fernandez Sophie  Charles-Delobel Chrystèle  Geldreich Angèle  Berthier Georges  Boyer Francine  Collonnier Cécile  Coué-Philippe Géraldine  Diolez Annick  Duplan Marie-Noëlle  Kebdani Naïma  Romaniuk Marcel  Feinberg Max  Bertheau Yves
Institution:Laboratoire Méthodologies de la Détection des OGM, Unite PMDV, INRA Versailles Route de Saint-Cyr RD 10, 78026 Versailles, France.
Abstract:A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.
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