Assessment of genetically engineered events in heat-treated and non-treated samples using droplet digital PCR and real-time quantitative PCR

Abstract

Regulations and labeling laws established by many countries for genetically engineered (GE) events necessitate the development of effective testing methods. Both real-time quantitative PCR (RT-qPCR) and digital PCR (dPCR) have been used for the detection and quantification of GE events. The advantage of digital PCR is that there is no need to use either a standard curve or reference materials to perform the analysis. In the case of RT-qPCR, incorrect results may be obtained if the standard curve does not reflect an amplification efficiency between 90 and 110%. DNA of high purity is required for obtaining accurate results from both PCR platforms. Food processing results in the degradation of DNA, which may affect PCR quantification of GE traits. In this study, the simulated effect of processed vs. non-processed samples on the testing of GE traits was evaluated using both droplet digital PCR (ddPCR) and RT-qPCR. Ground canola and soybean samples were heat-treated in boiling water for 15, 30 and 60 min in order to cause DNA degradation to determine the impact on quantification using both ddPCR and RT-qPCR. The measured ddPCR concentrations were similar to the gravimetric fortification by weight for three of the four GE events tested for both treated and non-treated samples. For one event, the concentrations obtained for treated samples were higher by roughly a factor of two than the expected 0.1% and 1% by weight target values. RT-qPCR assays gave similar results to those obtained using ddPCR.