title: Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.
abstract: Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.
authors.affiliation: †Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: David
authors.initials: D
authors.lastname: Dobnik
authors.affiliation: ‡Section of Virology, Norwegian Veterinary Institute, P.O. Box 750, Sentrum, 0106 Oslo, Norway.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Spilsberg
authors.affiliation: †Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.|§Josef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Bogožalec Košir
authors.affiliation: ‡Section of Virology, Norwegian Veterinary Institute, P.O. Box 750, Sentrum, 0106 Oslo, Norway.
authors.forename: Arne
authors.initials: A
authors.lastname: Holst-Jensen
authors.affiliation: †Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
mesh_terms: D005062:European Union; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics*; D015203:Reproducibility of Results; D003313:Zea mays / Q000235:genetics*