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.
Highlights
Modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of Genetically modified organisms (GMOs) as well as the worldwide area planted with biotech crops continues to increase steadily [1]
Available Digital Polymerase chain reaction (PCR) (dPCR) instruments seem to be suitable for quantification of GMO events
Assays used for quantitative PCR (qPCR) can readily be transferred to dPCR; some optimization of the primer and probe concentrations might potentially improve the overall assay performance
Summary
Modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of GMOs as well as the worldwide area planted with biotech crops continues to increase steadily [1]. Sample partitioning allows reliable detection of rare targets in a high background of non-target DNA, which is important for GMO analysis, where a transgene (GMO event) might be present at much lower concentrations than the reference gene (endogene) Another important advantage of dPCR is its lower sensitivity to PCR inhibitors. Such an approach was reported for maize [54] and soybean [53] This is not common practice in the EU enforcement laboratories, such an approach can be used for testing legal compliance of EU-authorized GMOs. this is not common practice in the EU enforcement laboratories, such an approach can be used for testing legal compliance of EU-authorized GMOs Another approach, amplitude-based multiplexing, allows quantification of four individual targets simultaneously in one reaction, but has some limitations in terms of specificity when highly concentrated DNA samples are used, because of the possible presence of droplets with intermediate fluorescence [56]
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