Construction of a reference plasmid containing ten targets for the detection of genetically modified crops

Standard reference molecules play a significant role for the detection of genetically modified (GM) crops and products. The newest reference molecules should catch up with the rapid development of GM crops in the world. In this work, a reference plasmid containing ten targets from GM soybean, maize and cotton was constructed on the basis of the pTLE8 harboring eight targets only from GM soybean and cotton. Three target segments of the Bt176 event-specific 3'-junction (Bt176G3'), MON810 event-specific 3'-junction (MON810G3') and the endogenous maize Hmg genes, were fused into the 890 bp fragment by overlap extension PCR. The CP4 EPSPS gene in the plasmid pTLE8 previously constructed in our laboratory was replaced with above fusion fragment, thus generating a new plasmid pTLH10 containing ten target genes from GM soybean, maize and cotton. The PCR efficiencies with pTLH10 as a calibrator ranged from 93.3% to 99.9% for the standard curves of the Bt176G3', MON810G3' and Hmg genes. The standard deviation (SD) values of repeatability were from 0.04 to 0.8 for three different days and from 0.12 to 1.14 for one day, respectively. These results indicated that the reference plasmid constructed in this work is also suitable for the identification of GM maize, and would be an important tool to establish a feasible identification management for various GM crops components.