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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

Surekha Karudapuram, Ph.D. and David Batey, Ph.D.
MJ Research, Inc., South San Francisco, CA


Abstract
We present a real-time quantitative PCR (qPCR) protocol for the detection and quantitation of soy genetically modified to be resistant to the herbicide glyphosate. The assay utilizes SYBR Green I (SGI) detection of amplicons of the genetically modified organism (GMO) specific sequences as well as a common soy gene as a reference. The assay can detect GMO materials down to 0.5% (w/w) concentration in foods. The use of SGI for detection makes this protocol readily adaptable for the analysis of other food crops for the presence of GMO material.


Introduction
A wide variety of food crops have been genetically engineered to contain beneficial traits such as herbicide resistance, disease resistance and insect resistance. Accurate and reliable detection of genetically modified organisms (GMOs) in foods is becoming increasingly important as the demand for labeling of GMO-containing foods increases.

Perhaps the most widely utilized modification that confers herbicide resistance is that which confers resistance to glyphosate [N- (phosphonomethyl)glycine]. Glyphosate is a competititive inhibitor of enolpyruval-shikimate-3-phosphate synthase (EPSPS), which is required for the production of aromatic amino acids by plants.1

To create glyphosate resistance, plants are transformed with a glyphosate tolerant version of the EPSPS gene from strain CP4 of the soil bacterium Agrobacterium sp.2, 3 Expression of the CP4 EPSPS in such modified plants confers resistance to glyphosate, and thus allows the application of glyphosate to fields of the growing GMO crop, e.g., Roundup Ready soybeans.

Because GMOs contain specific and well-defined nucleic acid sequences, they are well suited for detection by the polymerase chain reaction (PCR). Indeed, PCR has been shown to effectively detect GMO material, using an agarose gel analysis of the reaction products.4, 5

In this Application Note, we extend the PCR detection of GMOs by presenting a real-time qPCR protocol using SYBR Green I (SGI) performed on the DNA Engine Opticon 2 continuous fluorescence detection system. DNA was isolated from reference materials containing known proportions of GMO soy, as well as from soy products obtained from a local supermarket. Control primers that amplified a portion of the soy lectin gene were used to normalize the amount of soy DNA in each sample. A second set of primers that amplify a portion of the glyphosate-tolerant EPSPS gene were used for GMO detection.

The threshold cycle [C(t)] of a reaction is defined as the point when a reaction signal just rises over the background signal level. Comparing the relative C(t)s of lectin and EPSPS reactions from the GMO concentration reference series allowed the construction of a standard curve, from which the amount of GMO materials in unknown foodstuffs could be determined down to approximately 0.5% (w/w) concentration.


Materials and Methods
Isolation of DNA from Reference Standards and Food Samples
Dried soybean powder containing mass fractions of 0% to 5% Roundup Ready soybean (RTC Corporation, IRMM-41050-56) were used as reference standards. The reference standards were prepared by the manufacturer by mixing dried non-genetically modified and Roundup Ready soybean powders. The following foods were obtained from a supermarket for testing: soy burger, a soy dessert and two brands of soy flour.

The dry samples, the reference standards and the soy flour were used directly in the DNA isolation while wet samples such as the soy burger and the soy dessert were mashed to a fine, homogeneous paste with a mortar and pestle. For each standard and sample, the amount of starting material used for DNA isolation is shown in Table 3.

The DNeasy Plant Mini Kit (Qiagen, 69104) was used for isolation of DNA from reference standards and food samples. DNA was extracted from the different samples as described in the kit protocol. DNA concentration was determined by spectrophotometry and the quality verified by agarose gel electrophoresis on a 1% TBE gel. On average, we were able to obtain between 12g of DNA from each sample material.

Reactions
DyNAzyme II DNA polymerase* was from Finnzymes (F-503L) and SGI was from Molecular Probes (P-7589). Reaction components were assembled in low-profile microplates (MJ Research, MLL-9651) and sealed with ultra-clear strip caps (MJ Research, TCS-0803). Volumes of individual components and final reaction concentrations are listed in Table 1.

SGI was obtained at 10,000X concentration and was diluted to a 10X working concentration with 0.1X TE buffer, pH 8.0. A 10X SGI solution is defined as one giving 0.400.01 OD when measured at 495nm.

Primers for soy lectin and EPSPS were ordered from Sigma Genosys (The Woodlands, Texas). The primers used for amplification of the soy lectin gene generated a 318bp amplicon. Sequences of the lectin primers were: forward, 5-GAC-GCT-ATT-GTG-ACC-TCC-TC-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 product with the EPSPS primers, the ΔC(t) value was designated as not detected. Next, a standard curve was generated by plotting the log %GMO concentration of the reference standards against the ΔC(t) value (see Figure 1B). In order to calculate the %GMO content of an unknown food sample from the standard curve, the Excel Trend function was used. In the Trend function, the following values were specified for the standards: log %GMO (the known y values), ΔC(t) (the known xs) and the ΔC(t) of the unknown food sample for the new x. The value being generated from the formula is the log %GMO of the unknown. The %GMO soy content of the unknown sample was then determined from the formula 10 (log%GMO).

Thermal denaturation profiles of all reactions were examined to verify the presence of the lectin amplicon (in all soy) and the EPSPS amplicon (GMO soy specific).


Results
A standard curve generated from the soy reference standards and the EPSPS primers is shown in Figure 1A. The linear range of the curve extended from 5% mass fraction of GMO soy standard down to 0.5% mass fraction of GMO soy standard, with the square of the correlation coefficient (r2) equal to 0.992. The average C(t) values for duplicate reactions of each sample DNA with the lectin primers and the EPSPS primers are listed in Table 3. The amount of GMO soy was normalized to the amount of total soy present by calculating the ΔC(t) value as described in the Materials and Methods section.

The standard curve generated by plotting the log %GMO of the standards against the ΔC(t) values of the standards is shown in Figure 1B. The %GMO soy content of an unknown sample was then calculated from the Trend line of the ΔC(t) values for the known GMO concentration standards. As can be seen in Table 4, the soy dessert was found to contain 4.4% GMO soy. Since the ΔC(t) value for the soy burger was outside the range of values for the standards (more than that of the 5% standard), the GMO content was calculated from the trend as ~10.32%. Note that for both food samples the amount of GMO soy being calculated using the ΔC(t) value represents the amount of GMO soy relative to the total amount of soy present in the sample.

The results of the melting curve analysis are presented in Figures 2 and 3. Plotting the negative first derivative shows the melting point (Tm) as a single peak at approximately 88C for reactions with the lectin primers and 92C for reactions with EPSPS primers (Figure 2). For all DNA samples, a single peak at 88C confirmed the presence of soy (Table 5). Among the food samples tested, only the DNA from the burger and soy dessert samples generated a distinct peak at 92C, consistent with the presence of GMO soy (Figure 2 and Table 4).

To eliminate the possibility that the soy flour samples failed to generate the epsps amplicon due to an inhibition of the PCR by some component of the isolated DNA, control reactions with internal standards were run in parallel with the samples being analyzed. Each food sample was spiked with 20mg of the 5% GMO reference standard. DNA was then isolated from the spiked sample and amplified in reactions with the lectin primers and the EPSPS primers. Comparison of the melting curves of the spiked and nonspiked soy flour samples show that the EPSPS-specific peak is present only in the spiked samples (Data not shown and Table 5) ruling out a false-negative result. It should be noted that the soy flour samples that are negative for the EPSPS gene might still contain other types of GMOs that were not being tested in this assay.

To confirm that the GMO-positive food samples did contain the EPSPS gene, the amplified PCR products from reactions with the EPSPS primers were sequenced. As expected, a BLAST search with the obtained DNA sequence showed 100% concordance with the sequence of the glyphosate tolerant version of the cp4 epsps gene from Agrobacterium tumifaciens (GenBank Accession AF464188). The reaction products were also examined on an agarose gel. The band pattern for all reactions was in complete agreement with the results of the real-time qPCR data (not shown).


Discussion
In this study, we have developed a real-time qPCR protocol for the DNA Engine Opticon 2 system that can be used to detect and quantify genetically modified soy. The GMO detection assay presented here has several features that make it attractive for rapid processing of multiple samples. The amount of required starting material is quite small. We have found that between 50 to 100mg of a given food sample yields more than enough DNA for testing. Detection with SGI can be used for any DNA amplicon, allowing this protocol to be easily adapted for detection of GMOs in other crops such as maize, peanut and rice.


References
1. Schonbrunn, E., Eschenburg, S., Shuttleworth, W.A., Schloss, J.V., Amrhein, N., Evans, J.N.S. and Kabsch, W. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic details. Proc Natl Acad Sci 98(4):1376-1380 (2001).

2. Padgette, S.R., Biest Re, D., Gasser, C.S., Eichholtz, D.A., Frazier, R.B., Hironaka, C.M., Levine, E.B., Shah, D.M., Fraley, R.T. and Kishore, G.M. Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. Journal of Biological Chemistry 266(33):22364-22369 (1991).

3. Barry, G., Kishore, G., Padgette, S. et al. Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. Curr Topics Plant Physiol 7:139-145 (1992).

4. Ahmed, F.E. Detection of genetically modified organisms in foods. Trends Biotechnol 20(5):215-223 (2002).

5. Tengel. C., Schler, P., Setzke, E., Balles, J. and Sprenger-Hauels, M. PCR-based detection of genetically modified soybean and maize in raw and highly processed foodstuffs. BioTechniques 31(2):426-429 (2001).

6. Amutan, M. and Batey, D. Real-time quantification of genomic DNA using DyNAzyme II DNA polymerase and SYBR Green I dye. Application Note Vol. 1, No. 1. MJ Research, Inc.


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