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

Abstract

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.

Keywords: SYBR Green I; TaqMan-probe; droplet digital PCR (ddPCR); forage; genetically modified (GM); real-time PCR (qPRC).

FIGURE 1

Frame of transgenic elements in the event 10 ryegrass genome (A). Location of the designed construct-specific primer pairs and probe targeting the transgene are in gray color. The transcriptional direction is indicated with arrow, and the perennial ryegrass rubisco promoter, 1SST-6G-FFT fusion protein gene, and FT4 terminator sequences are shown with dark green, blue, and red arrows, respectively. in black dotted rectangles possible location for the construct specific primers and probe. For designing the SST-FFT fusion protein gene, the terminal codon (TAG) of the perennial ryegrass SST gene was removed. GC content distribution (B) of 1SST-6G-FFT fusion protein gene in blue, compare with rice genic (a), rice genome average (b) and rice intergenic (c) in green. Red dotted lines showed a 200 base-pairs window where the set of primers and probe are located.

FIGURE 2

Detection and quantification of 1SST-6G-FFT construct, using qPCR with SYBR Green I (A) and fluorescent probe (B). In probe-base assay the target construct (1SST-6G-FFT, FAM) is shown in blue and the reference gene (LpCul4, HEX) in green. Y-axis shows relative fluorescence unit (RFU), and the X-axis denotes PCR cycle number.

FIGURE 3

Detection and quantification of 1SST-6G-FFT construct (FAM in blue) using ddPCR. (A) 1D fluorescence amplitude plot, where set threshold is shown with a pink line, blue dots indicate presence of the 1SST-6G-FFT sequence in the droplet, and gray dots indicate absence of the sequence. (B) Ratio of 1SST-6G-FFT construct and LpCul4. UT and T stand for untransformed and transformed, respectively. Error bars indicate the Poisson 95% confidence intervals for each measurement.

FIGURE 4

Comparison of qPCR and ddPCR in all agricultural commodities. Histograms indicate the average relative copy number in each tissue (left scale). Lines show the trend of the variation (CV%) of the qPCR and ddPCR assays (right scale).

FIGURE 5

Limit of detection and limit of quantification of 1SST-6G-FFT construct (FAM in blue), and LpCul4 (HEX in green) as reference gene using droplet digital PCR. Blue and green plots indicate the concentration of positive droplets (counts/μL; Y-axis on the left side) for the 1SST-6G-FFT and LpCul4 sequences, respectively, and the average concentration is shown the left side of the plot. Orange plots show the copy number ratio of 1SST-6G-FFT (Y-axis on the right side), which was calculated through the average concentration of FAM-positive droplets divided by HEX-positive droplets. Error bars indicate the Poisson 95% confidence intervals for each measurement.