Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the 3′-transgene integration sequence

Abstract

An event-specific detection method was developed based on the flanking sequence of an exogenous integrant in the transgenic maize MON863 which contains cry3Bb1 gene expressing a Bacillus thuringiensis Cry3Bb1 protein that is selectively toxic to a maize root worm pathogen. The 3′-integration junction between host plant DNA and integrated DNA of transgenic MON863 maize was isolated using thermal asymmetric interlaced (TAIL)-PCR. The event-specific primers and TaqMan probe were designed based upon the isolated 3′-integration junction sequence, and qualitative and quantitative PCR systems were established employing these designed primers and probe. In this system, the limit of detection of the qualitative PCR assay was estimated to be 40 initial haploid copies. The limit of quantitation of the quantitative PCR assay in authentic MON863 maize seeds was estimated to be approximately 80 haploid copies. GM MON863 contents were also quantified relative to endogenous maize starch synthase IIb ( zSSIIb) gene DNA, and the results were expressed as the percentage of genetically modified MON863 maize DNA relative to the total content of maize DNA. All the results indicated that the established MON863 event-specific qualitative and quantitative PCR detection system based on the 3′-integration junction was reliable, sensitive and accurate.