Development of multiplex and construct specific PCR assay for detection of cry2Ab transgene in genetically modified...

An efficient detection system for trait validation and screening of GMOs is a much sought after procedure, which could also help in regulatory compliance. Currently, in India, a number of cry2Ab transgene carrying GM crops are undergoing field trial i.e., MON15985 cotton, Bt rice, Bt okra, Bt corn, Bt brinjal, Bt potato and Bt tomato. In this study, we report a qualitative assay for detection for cry2Ab (326 bp). Further, the amplification compatibility with promoter, p35S (195 bp), terminator, t-nos (180 bp) and marker gene, npt II ( 215 bp) was also confirmed using Bt cotton event MON15985 as reference material. The detection sensitivity was 0.1% that is far below the requirement of the stringent European Union (EU) regulations of 0.9%. The target DNA when spiked with either MECH-12 (cry1Ac), RR-soya (epsps) or MON-810 (cry1Ab) showed no inhibitory effect on cry2Ab detection. Moreover, the cry2Ab specific transgene construct (1.9 kb) was amplified and its identity confirmed by a nested PCR. Hence, a comprehensive multiplex PCR method for detection of cry2Ab gene in a GM crop/products was established. This is possibly a first report showing concurrent amplification of cry2Ab transgene, promoter, terminator and marker gene.