APPLICATIONS OF THE INVADER ® ASSAY FOR FOOD AUTHENTICATION Ana Ortolá-Vidal 1 , Angus Knight 1 , Glen Donald 2 and Ro

1. APPLICATIONS OF THE INVADER® ASSAY FOR FOOD AUTHENTICATION Ana Ortolá-Vidal1, Angus Knight1, Glen Donald2 and Robert Roeven2 1. Leatherhead Food RA, Randalls Road, Leatherhead, Surrey, UK 2. Third Wave Technologies, 502 South Rosa Road, Madison, USA INTRODUCTION At present DNA-based methods for determining food authenticity are based on the polymerase chain reaction (PCR). Although PCR is an extremely sensitive technology and a versatile and useful research tool, it is difficult to use for quantitative applications. The Invader® technology is an alternative direct detection technique that offers quantitative analysis with potential for on-site analysis. In contrast to PCR, the Invader assay relies on signal amplification rather than sequence amplification, and is an inherently quantitative analytical approach. This study has demonstrated the application of this technology for the sensitive and quantitative detection of genetically modified soya. RESULTS AIM To determine the detection limit of the Invader assay for its applications in the detection of GM soya. INVADER TECHNOLOGY A specific ‘Invader’ oligonucleotide, upstream from the cleavage site, and a partially overlapping downstream probe form a specific structure when bound to the complementary target DNA sequence (Figure 1). This structure is recognised and cut at a specific site by Cleavase enzymes, resulting in release of the 5’ arm of the probe oligonucleotide. This fragment then acts as the ‘Invader’ oligonucleotide in a secondary reaction, resulting in specific cleavage of the signal probe by Cleavase enzymes. Fluorescence signal is generated when the signal probe, labelled with dye molecules capable of fluorescence resonance energy transfer (FRET), is cleaved. The fluorescence signal generated may be detected using a fluorescence plate reader. Biplex Format More recently, the Invader assay has been converted to a biplex format. Here the internal control and the transgenic probe each have a unique 5’ flap sequence complementary to their own unique FRET oligos with two spectrally separate dyes, thus allowing simultaneous detection of GM DNA and internal control in the same microtitre plate well. • Limit of Detection The fluorescence values obtained were plotted in graphs that show the fluorescence signal generated by the total DNA (adh) and the GM DNA (CaMV). Figures 2 and 3 show results for incubation times of 4 h and 8 h, respectively. FIGURE 2 FIGURE 3 • Linearity Assessment Figures 4-7 represent the graphs plotted to assess the linearity of the GM DNA fluorescence signal, at incubation times of 4 h and 8 h. FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 • Biplex Format Evaluation Figures 8 and 9 illustrate the biplex invader assays for CaMV and nos detection, respectively nos detection in corn and soya CaMV detection in corn and soya 4.50 3.00 tubulin tubulin 4.00 nos CaMV 2.50 3.50 3.00 fold-over-0 fold-over-0 FIGURE 1 2.00 2.50 2.00 MATERIALS AND METHOD Mixes of GM- and non-GM soya at different % were analysed for GM DNA detection. Two probes were developed for the assay: the transgenic probe, specific for the CaMV 35-S promoter sequence present in GM DNA, and the internal control probe, specific for the soya alcohol dehydrogenase (adh) gene, which functions as a measure of the total amount of DNA in the reaction. Biplex Format GM- and non-GM maize and soya samples were tested with two biplex assays for GM DNA detection. The first probe, detected by Redmond red, is specific for either the CaMV 35-S promoter sequence or the nopaline synthase (nos) terminator. The second probe, detected by fluorescein, is specific for -tubulin and functions as a measure of the total amount of DNA in the reaction. 1.50 1.50 1.00 1.00 control control Soya 1 Soya 2 Corn 1 Corn 2 Corn 3 Corn 4 control control Soya 1 Soya 2 Corn 1 Corn 2 Corn 3 Corn 4 CaMV tubulin nos tubulin sample sample FIGURE 8 FIGURE 9 • DISCUSSION • After 4 h the limit of detection of the assay was found to be 1.5 ng of GM DNA in 200 ng of sample (approximately 0.75% w / w). The values obtained after 4 h showed good linearity • At longer incubation periods (8 h), the assay was no longer linear at the higher values, where the plateau of the curve was reached, whereas the linearity increased towards the lower end of the range. • In conclusion, the Invader assay showed good quantitative potential compared with alternative techniques, such as PCR. • The biplex format Invader assay gave accurate qualitative results for both the CamV and nos detection, and will be further evaluated for quantitative detection. ACKNOWLEDGMENT This project was carried out with the financial support of the UK Food Standards Agency REFERENCES 1. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes, Lyamichev V. et al., Nature Biotechnology, 1999, Vol. 17, page 292. 2. Clinical, Genetic and Pharmocogenetic Applications of the Invader Assay, Kwiatkowski R. W. et al., Molecular Diagnosis, 1999, Vol. 4.