Development and Application of Droplet Digital PCR Tools for the Detection of Transgenes in Pastures and Pasture-Based Products

Implementation of molecular biotechnology, such as transgenic technologies, in forage species can improve agricultural profitability through achievement of higher productivity, better use of resources such as soil nutrients, water, or light, and reduced environmental impact. Development of detection and quantification techniques for genetically modified plants are necessary to comply with traceability and labeling requirements prior to regulatory approval for release. Real-time PCR has been the standard method used for detection and quantification of genetically modified events, and droplet digital PCR is a recent alternative technology that offers a higher accuracy. Evaluation of both technologies was performed using a transgenic high-energy forage grass as a case study. Two methods for detection and quantification of the transgenic cassette, containing modified fructan biosynthesis genes, and a selectable marker gene, hygromycin B phosphotransferase used for transformation, were developed. Real-time PCR was assessed using two detection techniques, SYBR Green I and fluorescent probe-based methods. A range of different agricultural commodities were tested including fresh leaves, tillers, seeds, pollen, silage and hay, simulating a broad range of processed agricultural commodities that are relevant in the commercial use of genetically modified pastures. The real-time and droplet digital PCR methods were able to detect both exogenous constructs in all agricultural products. However, a higher sensitivity and repeatability in transgene detection was observed with the droplet digital PCR technology. Taking these results more broadly, it can be concluded that the droplet digital PCR technology provides the necessary resolution for quantitative analysis and detection, allowing absolute quantification of the target sequence at the required limits of detection across all jurisdictions globally. The information presented here provides guidance and resources for pasture-based biotechnology applications that are required to comply with traceability requirements.