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Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System

C-3 and reverse, 5-GAA-AGT-GTC-AAG-CTT-AAC-AGC-GAC-G-3.5 The primers used for amplification of the Roundup Ready-specific EPSPS gene generated a 356bp amplicon. Sequences of the EPSPS primers were: forward, 5-TGG-CGC-CCA-AAG-CTT-GCA-TGGC- 3 and reverse, 5-CCC-CAA-GTT-CCT-AAA-TCT-TCA-AGT-3.5 For both primer sets, the optimal denaturation and annealing temperatures were determined from denaturation and annealing gradient reactions (data not shown).

Following reaction assembly, the plates were transferred to the DNA Engine Opticon 2 fluorescence detection system (MJ Research) where cycling was performed in calculated mode according to the program listed in Table 2

Data Collection and Analysis
The C(t) line (cycle threshold) was set manually such that the line intersects the fluorescence traces at a point where the signals surpass background noise and begin to increase exponentially. This threshold is automatically applied to all wells and allows for the comparison of standards and samples at a point that provides the most consistent results.6

The %GMO soy content of the unknown food samples was calculated using a comparison of the difference in C(t) values for lectin and EPSPS amplification in an Excel spreadsheet. For every standard and sample, the C(t) value of the reaction with the lectin primers was subtracted from the C(t) value of the reaction with the EPSPS primers to generate a ΔC(t) value. The use of the ΔC(t) value assumes that the two reactions have similar efficiencies and that they proceed in a mutually independant manner since they are set up in separate wells. For those standards and samples that did not amplify a p
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