Detection and Quantitation of Genetically Modified Maize (Bt-176 Transgenic Maize) by Applying Ligation Detection Reaction and Universal Array Technology ROBERTA BORDONI,ALESSANDRA MEZZELANI,CLARISSA CONSOLANDI, ANDREA FROSINI,ERMANNO RIZZI,BIANCA CASTIGLIONI,CLAUDIA SALATI, NELSON MARMIROLI,ROSANGELA MARCHELLI,LUIGI ROSSI BERNARDI, CRISTINA BATTAGLIA, AND GIANLUCA DE BELLIS* Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche and Dipartimento di Scienze e Tecnologie Biomediche, Universita ` degli Studi di Milano, LITA, Via F.lli Cervi, 93 20090 Segrate (MI), Italy We have applied the ligation detection reaction (LDR) combined with a universal array approach to the detection and quantitation of the polymerase chain reaction (PCR) amplified cry1A(b) gene from Bt-176 transgenic maize. We demonstrated excellent specificity and high sensitivity. Down to 0.5 fmol (nearly 60 pg) of PCR amplified transgenic material was inequivocally detected with excellent linearity within the 0.1-2.0% range with respect to wild-type maize. We suggest the feasibility of extending the LDR/universal array format to detect in parallel several transgenic sequences that are being developed for food applications. KEYWORDS: Genetically modified organisms; GMOs; polymerase chain reaction; PCR; ligation detection reaction; LDR; universal array; Bt-176 transgenic maize INTRODUCTION Identification of genetically modified organisms (GMOs) in food is becoming an issue of great interest. The introduction in the market of GMO products raises social and ethical concerns, and the European Union has dictated several directives and regulations regarding their liberation and commercialization. This includes the Novel Food Regulation (258/97/CE) for labeling of GMO-containing food-stuff and the Directive 49/ 2000, which establishes that under 1% of approved GMOs, labeling is no longer necessary as long as their presence in the product is accidental or technically unavoidable (1). GMOs could be detected by either polymerase chain reaction (PCR), for direct detection of the transgenic DNA, or immunological methods, for detection of proteins coded by the transgenic sequences. Because of their higher reliability and sensitivity and the higher stability of DNA over proteins, PCR methods are usually preferred (2). PCR has been applied to the detection of FLAVR SAVR tomato (3), Roundup Ready soybean (4, 5), Bt-maize (6), and gbss-antisense transgene potatoes (7). Sequence confirmation of PCR amplicons by proper methods is mandatory to avoid false positives. With the increasing number of GMOs that are being developed for food application, the ability to detect several transgenes in a single reaction becomes an important feature of any detection method (8). Therefore, highly automated and parallel detection systems for the identification of GMO in foods are required. Recently microarray technology has been proposed for several applications in DNA analysis, allowing for the detection of different nucleic acid targets at once. Rudi et al. (9) recently proposed a microarray approach to GMO detection in food by multiplex PCR amplification, GMO specific primer extension, and hybridization of the resulting labeled templates to an oligonucleotide microarray. A different approach based on a universal array could be considered. Universal arrays consist of a set of synthetic oligonucleotides named ZipCodes (10) or Tags (11) of similar thermodynamic characteristics (melting temperature) but very different in sequence. Their sequences are totally unrelated to those under scrutiny. The ligation detection reaction has been proposed by Gerry et al. (10) in combination with a demultiplexing universal array as a powerful tool for sequence discrimination. This procedure requires the design of two adjacent probes specific for each target sequence. The discriminating oligo is 5′ fluorescently labeled, while the 5′ phosphorylated common probe brings a 3′ unique and artificial sequence named cZip- Code, which is complementary to a ZipCode sequence included within the universal array. These oligos hybridize consecutively along the template, and a thermostable DNA ligase joins their ends linking the fluorescent label with a specific cZipCode. This reaction can be cycled to gain in sensitivity. The resulting products are hybridized onto the universal array where the * To whom correspondence should be addressed. Telephone: +39022- 6422764. Telefax: +390226422770. E-mail: gianluca.debellis@itb.cnr.it. J. Agric. Food Chem. 2004, 52, 1049-1054 1049 10.1021/jf034871e CCC: $27.50 © 2004 American Chemical Society Published on Web 02/04/2004