Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays)

Abstract

We report the characterisation of the 3'-integration junction between host plant DNA and integrated gene-construct of the genetically modified Bt11 maize, and the successive development and first validation of a transformation event-specific quantitative TaqMan 5'-nuclease PCR assay for detection and quantitation of Bt11 maize on the LightCycler. The transgenic DNA in Bt11 is inserted in a tandem repeated DNA sequence motif of approximately 180 bp, and we discuss some of the problems this creates for design, development and validation of event-specific PCR assays. The limit of detection of the PCR assay was estimated to 10 initial template copies. The limit of quantitation in pure maize materials was estimated to approximately 40 template copies. In food samples containing high levels of exogenous DNA, the impact of this DNA was estimated to increase the limit of quantitation to approximately 100 initial template copies. Because of the low number of template copies required for detection and quantification, in combination with small size of the amplicon (~100 bp), the assay may be applied to analyses of raw materials as well as processed food and feed.