Droplet Digital™ PCR Works for GMO Quantification
News May 08, 2013
More than 60 countries representing 40 percent of the world’s population require labeling of food and feed when genetically modified organisms (GMOs) reach certain thresholds. Screening for and quantifying GMOs is essential to the integrity of this labeling policy.
“Droplet Digital PCR could replace or be a good alternative to qPCR, the current benchmark in GMO quantification,” said Dr. Dany Morisset, the paper’s lead author and a researcher atSlovenia’s National Institute of Biology. Dr. Morisset, in collaboration with the EU Reference Laboratory for GM Food and Feed (EU-RL GMFF), also coordinates an international R&D project to standardize screening methods for detecting GMOs in food and feed.
The paper showed that Droplet Digital PCR (ddPCR™) technology is more accurate and reliable than real-time quantitative PCR (qPCR) for quantifying GMOs, especially at low levels. Study authors also found that the ddPCR method meets international food standards of applicability and practicality.
qPCR has Drawbacks for Detecting GMOs
The most common technique for quantifying the presence of GMOs is qPCR, thanks to its accuracy and precision. However, according to Dr. Morisset, qPCR has several drawbacks. It is often unreliable and inaccurate when quantifying very small numbers of DNA targets or when those targets are part of complex matrices such as foods or feed that contain inhibitory substances.
A 2010 research study found that chamber digital PCR (cdPCR) delivered accurate quantification at low target copy number without the need for a standard curve. The matrix also did not inhibit cdPCR because it is an end-point assay and therefore its data are less affected by amplification efficiency. However, Dr. Morisset says its high costs make cdPCR impractical for real-world use.
The ddPCR System Meets or Exceeds International Recommendations for Performance Parameters
Dr. Morisset learned about Droplet Digital PCR technology, which was developed as an alternative to cdPCR with its easy workflow, low cost, and high throughput. Commercialized as the QX100™ Droplet Digital PCR system the ddPCR system provides thousands more partitions than in cdPCR, resulting in greater precision and per-sample costs that are up to 150 times less.
The Slovenian researchers analyzed food and feed matrices containing different percentages of a well-characterized GMO transgene. They found the ddPCR system’s performance parameters (precision, accuracy, sensitivity, and dynamic range) complied with the guidelines of the EU-RL GMFF 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 greater tolerance to inhibitors found in matrices such as wheat flour and feed.
ddPCR Technology is Practical for Everyday Lab Use
International food safety standards specify that new methods should be easy for labs to implement in terms of cost, time, and workflow.
In the authors’ hands, a ddPCR assay requires 190 minutes and a qPCR assay takes 160 minutes for the typical number of samples run in parallel in midsize GMO laboratories. However, due to the greater number of PCR reactions required per sample in the qPCR assay, the time and expense of the qPCR assay grows rapidly with increasing sample throughput. Droplet Digital PCR is also simpler to set up and involves less hands-on labor than qPCR.
Dr. Morisset’s findings reveal that ddPCR is a less expensive alternative to qPCR due to the lower number of reactions. Droplet Digital PCR capitalizes on its ability to duplex as opposed to qPCR’s traditional approach of performing separate assays for both control and transgene targets. The ddPCR assay also doesn’t require reactions for a standard curve or dilutions due to lower anticipated inhibition.
In a new study in cells, University of Illinois researchers have adapted CRISPR gene-editing technology to cause the cell’s internal machinery to skip over a small portion of a gene when transcribing it into a template for protein building. This gives researchers a way not only to eliminate a mutated gene sequence, but to influence how the gene is expressed and regulated.