Event-Specific Quantitative Detection of Nine Genetically Modified Maizes Using One Novel Standard Reference Molecule

摘要

With the development of genetically modified organism (GMO) detection techniques,the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection,and real-time PCR is the most effective and important method for GMO quantification.An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity.This study establishes the event-specific detection methods for TC1507 and CBH351 maizes.In addition,the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11,Bt176,GA21,MON810,MON863,NK603,and T25) were systematically optimized and developed.In these PCR assays,the fluorescent quencher,TAMRA,was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal.To overcome the difficulties in obtaining the certified reference materials of these GM maizes,one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene,zSSIIb,was constructed and used for quantitative analysis.The limits of detection of these methods were 20 copies for these different GM maizes,the limits of quantitation were about 20 copies,and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template.Furthermore,nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision.The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples,and the precision expressed as relative standard deviations was from 0.83 to 26.20%.All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.