Development and Validation of Duplex, Triplex, and Pentaplex Real- Time PCR Screening Assays for the Detection of Genetically Modified Organisms in Food and Feed Ingrid Huber,* ,† Annette Block, † Daniela Sebah, † Fre ́ de ́ ric Debode, ‡ Dany Morisset, § Lutz Grohmann, ⊥ Gilbert Berben, ‡ Dejan S ̌ tebih, § Mojca Milavec, § Jana Z ̌ el, § and Ulrich Busch † † Bavarian Health and Food Safety Authority (LGL), Veterinä rstrasse 2, D-85764 Oberschleissheim, Germany ‡ Walloon Agricultural Research Centre (CRA-W), Chausse ́ e de Namur 24, B-5030 Gembloux, Belgium § Department of Biotechnology and Systems Biology, National Institute of Biology (NIB), Vecna pot 111, SI-1000 Ljubljana, Slovenia ⊥ Federal Office of Consumer Protection and Food Safety (BVL), Mauerstrasse 39-42, 10117 Berlin, Germany * S Supporting Information ABSTRACT: Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis. KEYWORDS: real-time PCR, multiplex, GMO, screening, validation, LOD ■ INTRODUCTION During a typical genetically modified organism (GMO) analysis, a “screening“ is performed as an initial step in the DNA analysis, in which a minimum set of PCR tests (targeting specific genetic elements) should allow conclusions to be drawn on the absence/presence of as many as possible genetically modified (GM) events. Only in the case of positive screening results, a second step using event-specific PCR assays will then specifically identify and, if required by the legislation, accurately determine the content of the individual event(s) present in the sample. The advantage of such a screening-based approach is that a minimum set of screening assays is sufficient to cover a maximum number of GM events. 1-3 In this way, there is no need to perform a high number of event-specific tests, and thus significant time and costs can be saved. With the growing number of GM events released on the market worldwide and their increasing genetic diversity, 4,5 the number of screening tests to be carried out needs to be increased accordingly. The use of multiplex real-time PCR can significantly facilitate future screening processes and should be regarded as a modern tool for time- and cost-saving GMO analysis. 1 There are already several studies published on the development of multiplex real- time PCR assays for GMO testing (reviewed in ref 3), but until now, there is still no evidence that they have been applied in routine analysis. The aim of our study was to develop a pentaplex real-time PCR assay which can be easily implemented in routine analysis, covering as many GMOs as possible according to the screening table presented by Waiblinger et al. 6 Real-time (TaqMan) technology was used because of the added value in terms of sensitivity and specificity applying probes. To achieve the highest possible coverage of commercialized GMOs, we chose to target the 35S promoter region of the cauliflower mosaic virus (P-35S), the nopalin synthase gene terminator from Agrobacterium tumefaciens (T-nos), a construct containing a 6- enolpyruvylshikimate-3-phosphate-synthase gene from Agro- bacterium tumefaciens strain cp4 with an upstream sequence of the ctp2 chloroplast transit peptide from Arabidopsis thaliana (ctp-cp4-epsps), and the two herbicide resistance genes for phosphinothricin: Basta resistance gene from Streptomyces hygroscopicus (bar) and phosphinothricine acetyltransferase gene from Streptomyces viridochromogenes (pat). Additionally, as many laboratories are not equipped with apparatus enabling simultaneous detection of five different fluorescent dyes, a duplex and a triplex assay covering the same genetic elements were developed to extend the range of instrument application. The Bavarian Health and Food Safety Authority (LGL, Germany) developed the screening assays, which were transferred to the Walloon Agricultural Research Centre (CRA-W, Belgium) for interlaboratory robustness testing. The pentaplex method was also transferred to the National Received: June 6, 2013 Revised: August 22, 2013 Accepted: August 24, 2013 Published: August 24, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 10293 dx.doi.org/10.1021/jf402448y | J. Agric. Food Chem. 2013, 61, 10293-10301