Research Article Received: 2 July 2009 Revised: 17 February 2010 Accepted: 17 February 2010 Published online in Wiley Interscience: (www.interscience.wiley.com) DOI 10.1002/jsfa.3961 Quantitative detection method for Roundup Ready  soybean in food using duplex real-time PCR MGB chemistry Maria Cristina Samson, Mariolina Gull ` ı and Nelson Marmiroli ∗ Abstract BACKGROUND: Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non- authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. RESULTS: Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready  soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION: In this study, a duplex qRTPCR using TaqMan  minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products. c  2010 Society of Chemical Industry Keywords: duplex qRTPCR; GMO; LOD/LOQ; model processed food; TaqMan  MGB chemistry INTRODUCTION In an attempt to regulate the increasing consumption of genetically modified organism (GMO)-derived products, a uniform traceability system defining provisions for the documentation of the flow of GMO-derived commodities has been established in the European Community (Commission Regulation EC 258/97; EC 50/2000; EC 1829/2003; EC 1830/2003). 1 Data gained by this system build the basis for labeling that is no longer triggered just by a positive testing of the products for the presence of GMO materials. Reliable detection and quantification of GMOs in food and feed are mandatory in labeling products containing GM ingredients over a 0.9% threshold. The method of detecting GMOs based on DNA amplification is currently the method of choice because of the high stability of the DNA molecule even under the extreme conditions encountered during processing of some food products. 2–4 Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved GM lines. 5–9 Several multiplexing approaches are therefore in development to provide a cost-efficient solution. The real-time PCR TaqMan  chemistry (Applied Biosystems, Foster City, CA, USA) is one of the most commonly used for GMO detection. 10 Some authors 11,12 have described quantitative construct-specific multiplex methods using TaqMan  assay to quantify the amount of Roundup Ready  soybean (RRS) in different food products. Analogue studies have been presented on event-specific detection of RRS. 13,14 In the hope of improving the specificity of the hydrolyzation between the probe and the amplicon, minor groove binder (MGB)-tagged non-fluorescent quencher (NFQ) probes have been developed. 15 MGB is a small crescent-shaped molecule that fits snugly into the minor groove of duplex DNA. When the TaqMan  probe hybridizes, the MGB stabilizes annealing by folding into the minor groove of the DNA duplex created between the probe and the target sequence. Various fluorometric detection systems such as molecular beacon, 16 Scorpion 17 and SYBR Green 18 have also been developed. Recently, a strategy already successfully used in the detection and quantification of RRS in seed and flour has been presented; 19 however, the application of the TaqMan  MGB- NFQ chemistry in determining GMO in foods is still not available. Considering the large number of GM constructs expected in the future and the complexity of detecting GMOs in foods and ∗ Correspondence to: Nelson Marmiroli, Department of Environmental Sciences, Genetics and Environmental Biotechnology Section, University of Parma, viale GP Usberti 11/A, 43123 Parma, Italy. E-mail: nelson.marmiroli@unipr.it Department of Environmental Sciences, Genetics and Environmental Biotech- nology Section, University of Parma, 43123 Parma, Italy J Sci Food Agric (2010) www.soci.org c  2010 Society of Chemical Industry