Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples

Abstract

Real-time PCR is commonly used for quantification of genetically engineered (GE) materials. Digital PCR (dPCR) assays have an advantage over real-time quantitative PCR assays in terms of sensitivity and precision. For meaningful real-time PCR results, availability of reference materials is critical in order to generate standard curves, which are not required for dPCR. In this study, the RainDance RainDrop™ Digital PCR system was assessed for absolute quantification of unadulterated samples of canola and soybean containing low levels of OXY235 canola and DP305423 soybean. Amounts of DNA used (300 ng or 1000 ng DNA) for droplet digital PCR (ddPCR) had an effect on the occupancy of the reference genes (PCR positive droplet count); but the variation observed in occupancy did not affect quantification of the spiked samples. Combining the target and reference primers/probes in a single PCR (duplex ddPCR) was successfully used for quantification of 1%, 0.1% and 0.01% spiked samples. Successful quantification of 1%, 0.1% and 0.01% spiked samples was also achieved with real-time PCR using 100 ng DNA. Detection of 0.001% spiked DNA samples was obtained only with ddPCR for OXY235 canola and DP305423 soybean events. Use of hydrosheared DNA for ddPCR resulted in better separation of target and reference clusters from quenched droplets compared with heated DNA or non-treated DNA. Overall, repeatable and satisfactory results were achieved for the spiked samples using ddPCR.