ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 346 (2005) 90–100 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.08.004 Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modiWed organisms in foods C. Peano a,1,¤ , R. Bordoni a,1 , M. Gulli b , A. Mezzelani a , M.C. Samson b , G. De Bellis a , N. Marmiroli b a Institute of Biomedical Technologies, National Research Council, Via Fratelli Cervi 93, Segrate, Milano 20090, Italy b Department of Environmental Sciences, University of Parma, Section of Genetics and Environmental Biotechnologies, Viale delle Scienze 11/A, Parma 43100, Italy Received 4 May 2005 Available online 25 August 2005 Abstract A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in geneti- cally modi Wed soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the eYciency of all reactions. This multiplex PCR has constituted the basis for an eYcient plat- form for genetically modi Wed organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High speciWcity and sensitivity were obtained.  2005 Elsevier Inc. All rights reserved. Keywords: Multiplex PCR; GMO; Ligation detection reaction; Microarray; Food traceability Polymerase chain reaction (PCR) 2 is the analytical sys- tem most widely used in the detection of genetically modi- Wed organisms (GMOs) because of its high sensitivity and reliability [1]. Accurate tests for GMO material in human and animal foods are needed to both detect the presence of unauthorized GMOs and quantify the percentage compo- sition of GMO material. So far, 14 varieties of corn and soybean have been authorized by both the US Department of Agriculture and the European Commission (EC), and more than 100 are in the developmental phase. Moreover, the EC requires that foods in which >0.9% content derives from GMOs be speciWcally labeled as such [2]. Therefore, sensitive and speciWc PCR systems that simultaneously amplify multiple target genes [3] are considered advanta- geous [4]. Multiplex PCR is expected to save considerable time and eVort in GMO detection by decreasing the number of reac- tions required [5]. Several studies have shown multiplex PCR to be a rapid and convenient assay for GMO detec- tion [6]. Matsuoka et al. [7] developed a multiplex PCR sys- tem to distinguish Wve commercial lines of genetically modiWed maize. James et al. [3] described three qualitative multiplex PCR systems for soybean, maize, and canola. A commercially available kit, the Biosmart Allin 1.0 GMO Screening System (Promega, Madison, WI, USA), permits * Corresponding author. Fax: +0039 02 26422770. E-mail address: clelia.peano@itb.cnr.it (C. Peano). 1 C. Peano and R. Bordoni contributed equally to this research paper. 2 Abbreviations used: PCR, polymerase chain reaction; DNA, deoxyri- bonucleic acid; LDR, ligation detection reaction; UA, universal array; IRMM, Institute for Reference Materials Measurements; GMO, geneti- cally modiWed organism; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; bp, base pair; CTAB, cetyl-trimetil ammo- nium bromide; EC, European Commission; SSC, standard saline citrate.