Innovative Application of Fluorescent Microsphere Based Assay for Multiple GMO Detection
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Innovative Application of Fluorescent Microsphere Based Assay for Multiple GMO Detection Anna Fantozzi & Monica Ermolli & Massimiliano Marini & Branko Balla & Maddalena Querci & Guy Van den Eede
Received: 28 September 2007 / Accepted: 16 November 2007 / Published online: 11 January 2008 # Springer Science + Business Media, LLC 2007
Abstract An innovative multiple screening protocol allowing the detection of specific DNA sequences of p35S and epsps in GM soya flours simultaneously was developed. For the application of the Luminex xMAP technology, two different sets of fluorescent beads were cross-linked to the specific oligonucleotide probes previously amplified and labeled by polymerase chain reaction (PCR) in the presence of a biotinylated nucleotide. The detection of the amplification products bounded by the streptavidin–phycoeritrin conjugate was performed using the Luminex-100 instrument. The system allows identification of extremely low amounts of labeled targets as of 7.8 10−4 nmol of p35S antiprobe with an associated value of repeatability relative standard deviation (RSDr) equal to 2.8. Parameters such as repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ) were considered to evaluate the performances of the assay. The potentiality of the system described in this paper enables us to look forward to a multiple target assay that is able to simultaneously detect and eventually quantify tens of target sequences occurring A. Fantozzi : M. Ermolli (*) : M. Marini : B. Balla : M. Querci : G. Van den Eede Biotechnology & GMOs Unit, Institute for Health and Consumer Protection, DG-Joint Research Centre, European Commission, I-2120 Ispra (Va), Italy e-mail: [email protected] A. Fantozzi Institute of Biochemistry and Genetics, Centre for Biomedicine Department of Clinical-Biological Sciences (DKBW), University of Basel, CH-4058 Basel, Switzerland M. Marini Reference Materials Unit, Institute for Reference Materials and Measurements, European Commission, 2440 Geel, Belgium
within the same sample preparation. The present study describes the first application of a multitarget fluorescent microsphere-based assay for DNA genetically modified organism (GMO) detection. Keywords GMO detection . epsps . p35S . Multiple assays . Fluorescent beads
Introduction The need to develop innovative multitarget systems to assess the presence of genetically modified events in food and feed products for control purposes is still an open issue. Adventitious contamination of GM material in a free food background is difficult to avoid, but labeling and traceability of GM presence are required by the EU legislation starting from 0.9 (European Commission 2004; European Commission 2003, regulations 1829 and 1830). At present, polymerase chain reaction (PCR) and Real Time PCR (RTiPCR) are the most popular techniques used to detect and quantify the presence of GM events for labeling and control purposes (http://gmo-crl.jrc.it). To comply with the EU requirements, with the aim to gain the European author
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