Short communication Evaluation of polymerase chain reaction and DNA isolation protocols for detection of genetically modified soybean Cibele dos Santos Ferrari, Luciana Lehmkuhl Valente, Fa ´ bio Cristiano Angonesi Brod, Caroline Tagliari, Ernani Sebastia ˜ o Sant’Anna & Ana Carolina Maisonnave Arisi* Departamento de Cieˆncia e Tecnologia de Alimentos, Centro de Cieˆncias Agra´rias, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, Floriano´polis-SC 880034-001, Brazil (Received 02 March 2006; Accepted in revised form 28 June 2006) Keywords DNA isolation, genetically modified organism foods, polymerase chain reaction analysis, soybean products. Introduction DNA-based methods using polymerase chain reaction (PCR) have become widely applied to the detection of genetically modified organisms (GMO). Quality and purity of nucleic acids are some of the most critical factors for PCR analysis (Herman et al., 2003; Olexova´ et al., 2004). In order to obtain purified DNA free from inhibiting contaminants, suitable isolation methods should be applied (Anklam et al., 2002; Van Duijn et al., 2002). Although validation of GMO detection in different types of matrices is frequently discussed, it should be seen in relation to validation of DNA extraction methods rather than in relation to the PCR methods (Miraglia et al., 2004). Many DNA isolation methods use a cetyltrimethylammonium bromide (CTAB) extraction buffer, some of these methods have been considered efficient methods for a wide range of plant-derived foods, especially because of the good separation of polysaccharides from DNA (Anklam et al., 2002; Gryson et al., 2004; Olexova´ et al., 2004). The use of a suitable combination of different primer sets and the performance of adequate control experi- ments are prerequisites for very sensitive and accurate detection of GMO (James et al., 2003; Yamagushi et al., 2003). Although real time PCR methods for GMO quantification are available, they are a high cost analysis and qualitative PCR methods are still essential for screening the presence of GMO in food, mainly in developing countries (Oraby et al., 2005). Roundup Ready TM (RR) soybean is the first commer- cially available GM crop in Brazil. Cardarelli et al. (2005) and Greiner et al. (2005) demonstrated that food products sold in Brazil contained above 1% GM mater- ial, but none of these food products were appropriately labelled. There has been an increasing demand on testing laboratories to develop or adopt qualitative and quantitative methods to assure compliance of GM organ- isms labelling regulation (Cardarelli et al., 2005). In this study, three PCR primers pairs were selected according to the literature (Meyer & Jaccaud, 1997; Vollenhofer et al., 1999; Ko¨ppel et al., 1997) in order to compare the specific detection of RR soybean DNA. The DNA was extracted from soybean and food samples by three different protocols derived from a CTAB method, which was chosen because of its low cost and high feasibility. Material and methods Samples The certified conventional soybean samples were ob- tained from Ecocert (Floriano´polis, Brazil) and RR soybean (Monsanto Company, St. Louis, MO, USA) samples were kindly provided by Brazilian farmers from Rio Grande do Sul state, Brazil. Genetically modified RR soybeans and non-GM soybean samples were grinded in a blender and sieved (50 mesh). By adequate mixing of these samples (1:10), standard mixtures containing 0%, 0.001%, 0.01%, 0.1%, 1% and 10% w/w RR soybean were prepared in-house. After mixing, the samples were stored at )20 °C. The soy products (defatted soyflours, powdered soymilks, infant formulas containing 14% soy protein isolate and beverages containing 1% soy protein) were purchased from local supermarkets. Beverage samples were submitted to liophilisation before DNA isolation. *Correspondent: Fax +55 48 331 9943; e-mail arisi@cca.ufsc.br International Journal of Food Science and Technology 2007, 42, 1249–1255 1249 doi:10.1111/j.1365-2621.2006.01405.x Ó 2007 The Authors. Journal compilation Ó 2007 Institute of Food Science and Technology Trust Fund