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.

Semi-Quantification of GM Maize Using Ready-To-Use RTi-PCR Plates

“…For instance, the droplet digital PCR is promising in terms of cost effectiveness and reliability (Morisset et al 2013;Köppel et al 2015;Dobnik et al 2015;Lievens et al 2016). However, this technology is not yet available in most of testing laboratories, while PSP and the semi-quantification can be easily implemented with the standard GMO laboratory equipment.…”

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

“…Finally, an important aspect of routine analyses is cost-efficiency. Although analysis of GMO samples by simplex dPCR is more expensive, the use of multiplex approaches moves the scales in favour of dPCR [53, 54]. …”

§ 64 LFGB Kick-off Meeting zu „Anwendungspotenzial moderner Analysetechniken im Bereich Lebensmittel- und Futtermittelsicherheit und deren Authentizität“

“…In Deutschland konnte NGS schon erfolgreich im Rahmen einiger Ausbruchsaufklärungen zur Charakterisierung der Ausbruchsstämme angewendet werden (Mellmann et al 2011;Ruppitsch et al 2015). (Dobnik et al 2015), der Speziesdifferenzierung von pflanzlichen und tierischen Lebensmittel (Bucher and Köppel 2016;Cai et al 2014) …”