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

Abstract

The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstractThere are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

Cauliflower mosaic virus (CaMV) Biology, Management, and Relevance to GM Plant Detection for Sustainable Organic Agriculture

“…Given the increasing number and diversity of GMOs developed and the number of molecular biology technologies available in addition to PCR and qPCR, many different GMO detection methods have been developed (Broeders et al, 2012;Fraiture et al, 2015;Demeke and Dobnik, 2018) (for more information, see Supplementary Table 1 and references therein). Databases of GMO sequences and detection assays have also been compiled, facilitating the identification of an appropriate detection method or the development of a new technique for a given need (Dong et al, 2008;Petrillo et al, 2015;Wilkes et al, 2017).…”

Review of diseases and health management in zebrafish Danio rerio (Hamilton 1822) in research facilities

“…The two main benefits of ddPCR are that precise quantification of DNA in samples is not based on a standard curve and that it is independent of amplification efficiency. In addition, the partitioning of a PCR into tens of thousands of individual reactions dilutes the effects of inhibitors on the reaction (Demeke & Dobnik, 2018). This method is particularly suitable for environmental detection of DNA due to the highly variable concentrations of inhibitors found in these samples.…”

General methods for quantitative interpretation of results of digital variable-volume assays

“…The stability of polydisperse droplets prepared using this same technique was previous demonstrated 13 and the stability of the reagents used in this work has been previously noted. 21,22…”