These oligonucleotide primers were initially evaluated in silico using the program “wprimersearch” from the software “wEMBOSS” ( wEMBOSS). A standard 25 μl reaction volume is applied containing 0.625 U of DreamTaq™ DNA Polymerase BMS 777607 (Fermentas, CA, USA), 1× DreamTaq™
Buffer (Fermentas, CA, USA), 0.2 mM of dNTPs, 250 nM of each primer and 5 μl of DNA (10 ng/μl). The PCR program consisted of a single cycle of 10 min at 95 °C (initial denaturation) followed by 35 amplification cycles of 30 s at 95 °C (denaturation), 30 s at 60 °C (annealing) and 1 min at 72 °C (extension) and finishing by a single cycle of 10 min at 72 °C (final extension). The run was performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK). The PCR products were analysed by electrophoresis on a 1% agarose gel (INVITROGEN, CA, USA) (100 V, 400 mA, 60 min). The PCR products were purified using USB® ExoSAP-IT® PCR Product Cleanup (Affymetrix, CA, USA) according to the Selleckchem Androgen Receptor Antagonist manufacturers’ instructions. All sequencing reactions were performed on a Genetic Sequencer 3130XL
using the Big Dye Terminator Kit v3.1 (Applied Biosystems, CA, USA) ( Broeders et al., 2012c and Sambrook and Russell, 2001). The obtained sequences were analysed using the software “Nucleotide BLAST NCBI” ( ClustalW2, 2013 and Nucleotide BLAST NCBI, 2013). Considering the high diversity of genetic elements integrated in GM rices, our attention was focused on rice transformation vectors. Because of its presence in 30% of transgenic plants and, more particularly, in 65 and 53 peer reviewed publications on GM rices in 2011 and 2012, respectively, the pCAMBIA family vector was considered as a strategic target to detect a large spectrum of unauthorised GMOs (Ahmad et al., 2012, Kathuria et al., 2007, Komori
et al., 2007, Scopus, 2013 and Yu et al., 2012). The t35S pCAMBIA screening marker was thus developed to identify unauthorised GMOs containing a pCAMBIA family cassette. The t35S pCAMBIA-specific SYBR®Green screening method, generating an amplicon of 137 bp, was performed for integration in to the CoSYPS (Combinatory SYBR®Green qPCR Screening) for GMO detection, composed crotamiton of 18 SYBR®Green methods (RBCL, LEC, ADH, CRU, PLD, SAD1, GLU3, p35S, tNOS, pFMV, pNOS, t35S, Cry1Ab/Ac, Cry3Bb, pat, bar, epsps and CRT), which is able to run in a single 96-well plate (Barbau-Piednoir et al., 2010, Barbau-Piednoir et al., 2012a, Broeders et al., 2012a, Broeders et al., 2012b, Broeders et al., 2012c, European Union Reference Laboratory for GMFood, 2006, Mbongolo Mbella et al., 2011, Van den Bulcke et al., 2010, Vaïtilingom et al., 1999 and Yang et al., 2005a). The general structure of pCAMBIA vector is composed notably of p35S, tNOS and t35S elements (Cambia, Canberra, Australia).