Droplet-based digital PCR (dPCR) can quantify the copy numbers of transgenes in genetically modified organisms (GMOs) with better precision and at a lower cost than quantitative PCR (qPCR), according to a new study published this week in PLoS One (1).
Right now, qPCR is the standard method for evaluating GMOs, but it’s not ideal for food products, which contain molecular inhibitors that can interfere with DNA amplification, leading to inaccurate copy number estimates.
But some researchers believe this issue might not be as problematic in dPCR, which allows researchers to quantify DNA copy numbers by dividing a sample—through tiny droplets or physical chambers—into hundreds or thousands of separate reactions and then tallying the total number of amplifications by looking at each individual reaction. Although the concept of dPCR has been around for more than a decade, the cost and lack of validation of the technique has slowed adoption.
In the new study, Morisset and colleagues evaluated MON810 maize in a variety of different samples, using the endogenous gene HMG as an internal control. The group also tested samples containing MON810 maize that had been previously analyzed using qPCR.
Using Bio-Rad’s QX100, the only droplet-based digital PCR system available at the start of the study, Morisset’s group found that the system more accurately quantified the ratio of transgene to endogene copy number in a variety of certified reference materials when compared with qPCR. Also, “the dPCR reactions seem to be less sensitive to inhibitory substances than qPCR,” said Morisset.
Accounting for reagents, consumables, and labor, the researchers found that a 4 sample run using dPCR cost $20.90 per sample whereas qPCR cost $22.30 per sample. When more samples are run simultaneously, dPCR gets even more economical, Morisset said.
Both qPCR and droplet-based dPCR cost less than chamber-based dPCR systems, whose chips or plates range from $150-$400 each, according to Morisset. However, previous studies of Fluidigm’s BioMark system, which uses microfluidics to partition samples, have demonstrated that this platform could also be another alternative for GMO analysis (2,3).
Overall, Morisset was surprised by how easy it was to establish duplex reactions in the dPCR system. The platform required only one day of training. Although the instrument was not released in Europe when the study began, Morisset got early access by contacting the company, which then provided him with a loaner system. After trying the machine, he purchased one for his lab.
The group is watching other dPCR platforms from Life Technologies and RainDance with great interest, in the hope that competition will drive down prices. “In GMO quantification, I’m sure [dPCR] will become more and more important,” he added.
1. Morisset, D., D. Štebih, M. Milavec, K. Gruden, and J. Žel. 2013. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS ONE 8(5):e62583+.
2. Burns, M. J., A. M. Burrell, and C. A. Foy. 2010. The applicability of digital PCR for the assessment of detection limits in GMO analysis. European Food Research and Technology 231(3):353-362.
3. Corbisier, P., S. Bhat, L. Partis, V. R. D. R. Xie, and K. R. Emslie. 2010. Absolute quantification of genetically modified MON810 maize (zea mays l.) by digital polymerase chain reaction. Analytical and bioanalytical chemistry 396(6):2143-2150.