Multiplex PCR Detection of Four Events of GM Maize (Event 3272,LY038, MIR162, and MON88017)

Abstract

To date, a number of genetically modified (GM) crops have been planted in 23 countries. GM maize was grown on 35.2 million hectares (13% of the global biotech area) in 2007 [James, 2007]. Since the first approval of a commercial genetically modified organism (GMO) in 1996, many countries have approved commercial releases of various events of GM maize for food and feed. According to the GMO database of Agbios (http://www. agbios.com, Agriculture and Biotechnology Strategies Inc., Merrickville, Ontario), 47 events of GM maize including 18 stacked traits were developed and approved in several countries [James, 2007]. The KFDA (Korea Food and Drug Administration) has approved 28 events of GM maize. KFDA is additionally conducting safety assessments of several GM maize events (MIR162, MON89034, Event 3272, LY038, etc.). Korea first imported GM maize for food use (corn starch and syrup) in 2008. GM crops will continue to be developed in various countries and Korea may import more GMOs for food use in the near future. Therefore, detection methods should be continuously developed to monitor a labeling system for GM foods. In this study, an event-specific multiplex PCR detection method for four events of GM maize (Event 3272, LY038, MIR162, and MON88017) was devised. GM maize Event 3272 contains the thermostable alpha amylase (amy797E) gene from Thermococcus bacteria and the mannose-6-phosphate isomerase (pmi) gene from E. coli. GM maize LY038 contains the dihydrodipicolinate synthase (cordapA) gene from Corynebacterium glutamicum. GM maize MIR162 containing the vegetative insecticidal protein (vip3A) gene from Bacillus thuringiensis was developed to provide resistance to Lepidopteran species. GM maize MON88017 contains the Cry3Bb1 delta-endotoxin (cry3Bb1) gene for insect resistance from Bacillus thuringiensis subsp. kumamotoensis strain EG4691 and the 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) gene for glyphosate tolerance from Agrobacterium tumefaciens CP4. An event-specific PCR system designed based on the junction between the transgenic insert and the host DNA is regarded as the most specific approach [Heo et al., 2004]. Multiplex PCR detection methods have been developed for GM maize [Matsuoka et al., 2001; Heo et al., 2004; Hernandez et al., 2005; Onishi et al., 2005; Kim et al., 2006; Ahn et al., 2008], GM canola [Demeke et al., 2002; Kim et al., 2007], GM cotton [Kim et al., 2008], and mixed GM crops [James et al., 2003; Germini et al., 2004; Forte et al., 2005]. In this study, the multiplex PCR method was developed to efficiently monitor four events of GM maize (Event 3272, LY038, MIR162, and MON88017) in a single reaction using event-specific primers. Event 3272 and MIR162 were developed from Syngenta Seeds Inc. (Golden Valley, MN), and LY038 and MON88017 were developed from Monsanto Company (St. Louis, MO). Eleven different plants [soybean (Glycine max), corn (Zea mays), canola (Brassica napus), cotton (Gossypium hirsutum), rice (Oryza sativa), barley (Hordeum vulgare), buck wheat (Fagopyrum esculentum), wheat (Triticum aestivum), red-bean (Phaseolus angularis), radish (Raphanus sativus), and Chinese cabbage (Brassica rapa subsp. pekinensis)] were collected from Rural Development Association in Korea and the Department of Food Biotechnology at Kyung Hee University. Samples were finely ground in liquid nitrogen using a mortar and pestle. The DNeasy Plant Maxi Kit (Qiagen GmbH, Hilden, Germany) was used for DNA extraction according to the manufacturer’s instructions. PCR was carried out on a Mastercycler (Eppendorf, Hamburg, Germany). The 25-μL reaction volume contained 2.5 μL of 10× buffer (Applied Biosystems, Foster City, CA), 200 μM of each dNTP (Applied Biosystems), 1.5 mM of *Corresponding author Phone: 82-31-201-2660; Fax: 82-31-204-8116 E-mail: hykim@khu.ac.kr