Droplet digital polymerase chain reaction (ddPCR) has seen increasing applications in recent times, also in the analysis of genetically modified (GM) food and feed samples. While quantitative real-time PCR (qPCR) methods have been traditional mainstays till now, the applicability of ddPCR in routine analysis of GM food and feed has not yet been widely demonstrated. In this work, we applied ddPCR on selected GM-food and feed samples that were recently analyzed on the qPCR platform in inter-laboratory proficiency tests and showed good performance of the ddPCR method. Sometimes GM DNA at different transgene levels, useful as reference material is not readily available. Applying ddPCR, different concentrations of GM material, specifically transgene DNA at different levels (0.1e10%) useful as reference DNA, were generated from 100% non GM material and 100% transgene plant material respectively, and key performance parameters of the ddPCR assay evaluated. DdPCR performed well, indicating its suitability for the production of reference GM materials. In an expanded analysis, DNA extracted from a 100% GM reference soy plant (CV127) was appropriately diluted to low copy numbers and the absolute LOD95% determined at 2 copies (nominal value), comparing well with various published qPCR assays. In our inhibition studies, ddPCR showed a clear advantage over qPCR in SDS-inhibited samples, while its tolerance to other tested inhibitors was comparable with qPCR. Significantly, the qPCR assays demonstrated more asymmetrical amplification/inhibition with EDTA as inhibitor, with unequal inhibition in reference and transgene reactions, while inhibition was more symmetrical on the ddPCR platform. Finally, a panel of positive reference material with varying GM content from 0.1 to 10% were evaluated on the ddPCR platform and pertinent performance parameters assessed, namely, precision, accuracy and trueness of results, with good performance of the assay.
The analysis of genetically modified (GM) foods and feed has seen an increased surge in recent times. While the list of EUapproved GMO foods continues to grow, food and feed analysts are constantly evolving new detection (screening) and control strategies to keep up with the increasing analytical demands. Currently the gold standard in the detection and quantification of GMO events in food and feed is the quantitative real-time polymerase chain reaction (qPCR). Indeed several qPCR assays for GM analysis, with or without quantification, have been published to date (Holst-Jensen, 2009; Koppel, Bucher, Meuwly, € & Moor, 2014; Koppel, Zimmerli, € & Breitenmoser, 2010, European Union Reference Laboratory for GM Food and Feed (EURL-GMFF). While the majority of these assays are validated and applied as singleplex reactions, an increasing number of multiplex assays, detecting and quantifying simultaneously several GM events, have been described in recent years (Koppel, Velsen, Felderer, € & Bucher, 2012; Park, Kim, & Kim, 2015; Shin et al., 2013; Wang, Teng, Guan, Tian, & Wang, 2015). Although most qPCR approaches generate reasonably good results with acceptable levels of precision, they usually rely on the use of standard curves for the determination of unknown GMO content. With ddPCR on the other hand, GM content can be determined by measuring concomitantly the ratio of transgene relative to reference gene in the sample under investigation without using standard curves, either as two singleplex reactions in the same analytical run or in a combined duplex system.