Development of a multiplex Polymerase Chain Reaction method for specific detection of Genetically Modified Cotton Events MON 531 and MON 15985

The main objective of this study was to develop a reliable detection method for genetically modified cotton events MON 531 and MON 15985 by using multiplex PCR reaction technique. In this study we have used four primer pairs for the detection of individual gene segments present in transgene cassette of two GM cotton events which include CaMv35S promoter, Nos terminator, Cry1Ac and Npt II genes and one pair of primers for cotton endogenous reference gene, fiber specific acyl carrier protein (fsAcp). In addition, one event specific primers for MON 531 event and one construct specific primer pair specific for MON 15985 event were also analysed in the integration events. Duplex PCR and multiplex PCR methods were standardized. In duplex PCR individual gene segments were coamplified with fsAcp gene and the products were resolved by using high resolution agarose gel based on their amplicon sizes. Multiplex PCR was performed with the genomic DNA extracted from the GM seed and leaf samples of the two events. To test the limit of detection, the DNA mixture prepared from each of two GM cotton events and non GM cotton were used. The sensitivity of our assay was 0.03%. The multiplex PCR method reported in the present study is simple, cost effective and time saving. This method could be an effective tool for detection and evaluation of two specific GM cotton events.