ABSTRACT
The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range. In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters.
ABSTRACT
BACKGROUND: The number and variety of genetically modified organisms (GMOs) used globally for the production of food and feed, and potentially circulating in the European Union (EU), is constantly increasing. This implies an additional effort for the EU enforcement laboratories to optimize available resources, to contain costs and time. A well established approach for streamlining the analytical workflow is the introduction of a screening step, typically based on a smart set of real-time polymerase chain reaction (PCR) screening methods. The multiplexing strategy, allowing the detection of several screening elements simultaneously, is a further optimization of this step. RESULTS: In this study, we present the validation of a real-time PCR duplex assay for the pat and bar screening elements to be easily incorporated in the GMO diagnostic routine. We also provide a comparison between this method and the related singleplex and pre-spotted assays. CONCLUSION: Our results fully respect all the validation parameters suggested by the Minimum Performance Criteria of the European Network of GMO Laboratories. Furthermore, the duplex assay is equivalent in terms of performance compared to the other two methods, but it shows a higher overall flexibility and cost effectiveness. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
