Evaluation of RainDrop™ Digital PCR System for Absolute Quantification of Low Levels of Genetically Engineered OXY235 Canola Samples

Real-time PCR is commonly used for quantification of genetically engineered (GE) materials.  Digital PCR (dPCR) has an advantage over real-time quantitative PCR assay in terms of sensitivity and precision.  The availability of reference materials is critical for successful real-time quantitative PCR assays in order to generate a standard curve, which is not required for dPCR.  The RainDrop™ Digital PCR System was assessed for absolute quantification of low levels of GE OXY235 canola samples.  The average number of positive droplets per microliter for OXY235 target was 706, 62 and 7 for 1%, 0.1% and 0.01% spiked samples, respectively.  For the OXY235 canola samples spiked at 1%, 0.1% and 0.01% (GE: non-GE DNA), results close to the expected percentage values were obtained.  The use of duplex PCR (combining the target and reference primers/probes in a single reaction) also resulted in the expected percentage values for the spiked samples.  The use of either 300 ng or 1000 ng DNA for duplex PCR did not affect the results obtained.  Overall, consistent results were achieved for the replicated experiments.