Establishment of quantitative method for five events of genetically modified maize (Bt11, event176, GA21, MON810 and T25) by real-time QPCR

Abstract

In order to meet the implementated labeling requirement, this study focused on the development of real-time quantitative polymerase chain reaction (real-time QPCR) method to detect genetically modified maize (GM-maize). Primers and probes specific for inserted genes in Event176, Bt11 (Syngenta company), MON810, GA21 (Monsanto company) and T25 GM-maize (Aventis company) were designed and used to conduct the real-time QPCR assays. A plasmid containing both transgene and internal control gene targets on the same molecules for each GM-maize event was also constructed as quantitative reference molecules. Further, constructed plasmids and test samples were applied to validate this quantitative system. Results showed that the slope of the standard curve generated by serial dilution of constructed plasmids was in the range of -3.31--3.45 with a correlation of 0.99-1.0. Test samples spiked 1%, 2% and 5% GM-maize were quantitated by the method developed in the study. The mean values of 1%, 2% and 5% test sample were between 1.01%-1.23%, 2.00%-2.31% and 4.46%-5.55%, respectively. The standard deviation (S.D.) ranged from 0.05 to 0.46 with coefficient variance (C.V.) of 5%-15%. The limit of quantitation was 0.1% (w/w) for these GM-maize crops. In addition, proficiency test samples were analyzed by the method developed in this study and good results were obtained.