Development of Multiplex and Quantitative PCR Assay to Detect Genetically Modified Roundup Ready Soybean in Foods

WEI-YI WANG AND TONY J. FANG*

Department of Food Science, National Chung Hsing University, 250 Kuokuang Rd., Taichung City 402, Taiwan, R.O.C.

(Received: July 16, 2003; Accepted: December 15, 2004)

ABSTRACT

The objective of this study was to develop a qualitative detection method for genetically modified (GM) soybeans using the multiplex PCR technique. Potential applications for using the developed detection method to analyze GM material in soya and its products were also evaluated. Four primers for the detection of transferred genes in Roundup Ready soybean artificially synthesized for this study included 35sP (Cauliflower mosaic virus 35S promoter), nosT (Agrobacterium tumefaciens nopaline synthase terminator), and 35sP/CTP (Petunia hybrida EPSPS chloroplast transit peptide). In addition, Lec (Lectin) primers were used to detect soybean species specificity. The results showed using either 35sP or 35sP/CTP as a primer obtained a detection limit of 0.01% (w/w) and using either primer nosT or multiplex PCR with 35sP/CTP obtained a detection limit of 0.1% (w/w). Furthermore, the transferred genes in Roundup Ready soybeans were confirmed through the isolation and sequencing of their genes. By using the multiplex PCR method developed in this study, we detected GM soybean material in 14 of the 21 soybean products obtained from the open market for this study. In addition, samples with 20%, 10%, 5% and 1% GM-soya and 5% GM-soya standard were quantitatively analyzed with SYBR Green I, with an R2 of 0.9683 when regression analysis was applied. Our results showed that, in addition to readily detecting GM soybean material, the multiplex PCR method reduced detection time and costs. The multiplex PCR method proposed in this paper may offer a useful tool to detect and monitor GM foods.

Key words: multiplex PCR, genetically modified organism (GMO), Roundup Ready soybean