Problem: Food testing labs have traditionally used conventional PCR and quantitative real-time PCR (qPCR) to detect the presence of genetically modified organisms (GMOs) in food and feed. When quantification is required, GMO content in these samples is expressed in relative terms as the ratio of the quantity of the transgene, which is the nucleic acid fragment introduced in the host genome, to that of an endogene, a gene normally found in the host genome. Currently, more than 60 countries— representing 40 percent of the world’s total population—require food and feed to be labeled once they contain GMOs beyond a certain threshold. These countries have implemented strict regulations and policies pertaining to products containing GMOs, or any materials derived from GMOs. Thus, screening for and quantifying GMOs is critical to upholding the integrity of labeling policies.
The gold standard for analyzing the presence of nucleic acids in food and feed samples has been qPCR, because of its sensitivity, accuracy, precision, and specificity. However, qPCR also presents a unique set of challenges when it comes to GMO analysis. The technique can be unreliable and inaccurate when quantifying very small numbers of DNA targets. Accuracy becomes a greater concern when those DNA targets are part of complex matrices (e.g. seed-powder flour, corn flakes etc.) that contain substances that inhibit qPCR—a common occurrence in foods or feed.
Solution: Recent research1 has demonstrated that digital PCR technology is more accurate and reliable than qPCR for quantifying GMOs, especially for GMOs present at low levels. Digital PCR provides the absolute number of targets present in a sample, based on the principles of limiting dilution, endpoint PCR and Poisson statistics. Two approaches are used in commercially available dPCR systems. One approach, chamber digital PCR (cdPCR), relies on the partitioning of up to a few thousand individual reactions in microfluidic chambers. The second approach, droplet digital PCR, combines partitioning of the sample into several thousands of individual droplets in a water-oil emulsion, with the use of flow cytometry to count positive reactions. Droplet Digital PCR technology was developed as a more practical alternative to cdPCR; it features an easy workflow, lower per sample costs, and higher throughput. Commercialized by Bio-Rad Laboratories, Inc. as the QX100™ Droplet Digital PCR system, the ddPCR system provides thousands more partitions than cdPCR, resulting in greater precision and more affordable per-sample costs. While cdPCR does deliver accurate quantification at low target copy number, its high costs make it impractical for real-world use.
Dr. Dany Morriset at Slovenia’s National Institute of Biology conducted research that demonstrated ddPCR confers advantages relative to qPCR for GMO quantification in a number of areas. The team analyzed food and feed matrices containing different percentages of a well-characterized GMO transgene. The ddPCR system’s precision, accuracy, sensitivity, and dynamic range complied with the guidelines set by the European Union Reference Laboratory for GM Food & Feed and were comparable or superior to those for qPCR. Compared with the conventional qPCR assay, the ddPCR assay offered better accuracy at low target concentrations and exhibited greater tolerance to inhibitors found in matrices such as wheat flour and feed. International food safety standards specify that new methods should be easy for labs to implement in terms of cost, time, and workflow. The ddPCR technique showed strong improvements on all of these fronts. The technique is a less expensive alternative to qPCR due to the lower number of reactions and its duplex ability (i.e. interrogate two targets per well) as opposed to qPCR’s requirement of performing separate assays for both control and transgene targets. Thus, for food laboratories looking for higher throughput, lower cost and most importantly, more accurate screening methods for GMOs, droplet digital PCR is a viable option.
For more information on droplet digital PCR, visit http://www.bio-rad.com/QX200
1. Morisset D, Štebih D, Milavec M, Gruden K, Žel J (2013) Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR. PLoS ONE 8(5): e62583. doi:10.1371/journal.pone.0062583