Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1

Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes.