A novel trait-specific real-time PCR method enables quantification of genetically modified (GM) maize content in ground grain samples containing stacked GM maize

Stacked genetically modified (GM) maize is increasingly produced; thereby, current event-specific quantitative real-time polymerase chain reaction (qPCR) methods have led to the overestimation of GM organism (GMO) content compared with the actual weight/weight percentage of GM organism in maize samples. We developed a feasible qPCR method in which the GMO content is calculated based on the quantification of two herbicide-tolerant trait genes, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4 (cp4epsps) and phosphinothricin N-acetyl-transferase from Streptomyces viridochromogenes (pat) to quantify the GMO content in ground grain samples containing stacked GM maize. The GMO contents of two genes were quantified using a plasmid calibrant and summed for quantification of total GMO content. The trait-specific method revealed lower biases for examination of test samples containing stacked GM maize compared with the event-specific method. Our results clearly show that the trait-specific method is not only simple and cost-effective, but also useful in quantifying the GMO content in ground grain samples containing stacked GM maize, which are expected to be major events in the near future. The developed method would be the only feasible way to conduct the quantification of GMO content in the ground maize samples containing stacked GM maize for the verification of the labeling regulation.