However, most of the methods in these studies cannot easily be used in routine analysis by the enforcement laboratories: techniques are laborious and complex (fingerprinting by capillary electrophoresis, genomic DNA library via (unpredictable) see more restriction enzyme) with regard to a method exclusively based upon PCR, require a lengthy procedure with generally
multiple steps to get results, or present a lack of specificity, yield or data concerning the compatibility with a low amount of target. The aim of the present study is to supply an integrated approach to identify unauthorised GMOs: A first real-time PCR screening allows the detection of the terminator 35S (t35S) of the pCAMBIA family vectors to indicate the potential presence of unauthorised GMOs in food matrices (Fig. 3). Then, an appropriate DNA walking method, anchored on the sequence used for the screening followed by two semi-nested PCRs to identify the junction, confirms the GMO presence. Grains of transgenic Bt rice (O. sativa L. Japonica cv Ariete) and its wild-type (WT) were used in this study to develop the methodology ( Breitler et al., 2004). This transgenic rice was transformed
by A. tumefaciens with the binary vector pCAMBIA1300, which contains the synthetic Cry1B gene NVP-BGJ398 supplier from Bacillus thuringiensis conferring insect resistance. The Certified Reference Materials (CRM) in the form of seeds powders or genomic DNA (gDNA) were obtained from the American Oil Chemists’ Society and the Institute for Reference Materials and Measurements Rucaparib in vitro and were used to test the specificity ( AOCS, 2013 and IRMM (Institute for Reference Materials and Geel, 0000). These materials were characterised as previously described ( Broeders et al., 2012c). The list of all plant
material is shown in Table 1. Bt rice grains were ground to obtain a homogenous powder. DNA was extracted using a CTAB-based procedure (ISO 21571) in combination with the Genomic-tip20/G (QIAGEN, Hilden, Germany). This DNA extraction method, adapted from the EU-RL GMFF validated method, is composed of four main successive steps: (1) Extraction of proteins, polysaccharides and organic components, (2) Precipitation of DNA in presence of C-hexadecyl-Trimethyl-Ammonium-Bromide (CTAB), (3) Purification of DNA using a tip20 column and (4) Precipitation of DNA with isopropanol (European Union Reference Laboratory, 2006 and International Standard ISO 21571, 2005). DNA concentration was measured by spectrophotometry using the Nanodrop® 2000 (ThermoFisher, DE, USA) device and DNA purity was evaluated by the A260/A280 and A260/A230 ratios. DNA extraction, concentration and purity of CRMs were carried out as previously described (Broeders et al., 2012c). The oligonucleotide primers were designed to target the t35S sequence of the pCAMBIA vector (Fig. 1). To get universal oligonucleotide primers detecting all pCAMBIA vectors, all t35S pCAMBIA sequences were compared via the software “ClustalW2”.