Molecular toolbox for the identification of unknown genetically modified organisms |
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Authors: | Tom Ruttink Rolinde Demeyer Elke Van Gulck Bart Van Droogenbroeck Maddalena Querci Isabel Taverniers Marc De Loose |
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Institution: | (1) Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit—Product Quality and Innovation, Burg. Van Gansberghelaan 115, 9820 Merelbeke, Belgium;(2) KaHo Sint-Lieven, Departement Gent, Gebroeders Desmetstraat 1, 9000 Ghent, Belgium;(3) Molecular Biology and Genomics Unit, European Commission - Joint Research Center (JRC), Institute for Health and Consumer Protection, TP 331—via E. Fermi 2749, 21027 Ispra, Varese, Italy;(4) Department of Molecular Genetics, Ghent University, Technologiepark 927, 9052 Ghent, Belgium; |
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Abstract: | Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain
in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized
presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization
(unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic
elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are
tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence
is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative
event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events.
Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement
the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the
identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition,
we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally
generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO,
which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification
of a novel unknown event without prior knowledge of its transgene sequence. The results demonstrate that the new methods complement
routine screening procedures by providing direct conclusive evidence and may also be useful to resolve masking of unknown
events by known events. |
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