A rapid field-deployable transgenic event detection system in genetically modified (GM) cotton using annealing curve...

Genetically modified (GM) cotton events with insect resistance and herbicide tolerance traits have been commercialized worldwide. In India, insect resistant Bt cotton is commercially cultivated with the maximum acreage under MON15985 and MON531 GM events, whereas events such as COT102 and herbicide tolerant events MON88913, MON1445 are not approved. Henceforth, regular surveillance for unauthorized introduction of non-approved GM cotton events as well as for authentication of approved seeds is required through rapid methods facilitating in field or on-site inspections. Multiplex real-time loop-mediated isothermal amplification (LAMP) assays were developed to simultaneously detect MON531 and MON15985 events of approved Bt cotton along with cp4-epsps gene present in non-approved herbicide tolerant GM cotton events such as MON88913, MON1445, for detecting 0.1% of each GM target within 45 min. Another multiplex real-time LAMP for detection of MON88913 and COT102 events was developed with limit of detection up to 0.1%. The GM events were differentiated based on the annealing curve profiles. In combination with rapid DNA extraction protocol optimized herein without the use of any instrumentation, this method facilitated field deployable event detection due to portability of detection system. To our knowledge, multiplex real-time LAMP has been reported herein for the first time with application of multiple event-specific GM detection in cotton. This method specifically customized for event-specific multiplexing, enables simultaneous detection of multiple GM events of cotton, both authorized and unauthorized from the regulatory perspective in India. The developed method could be utilized globally for event detection based on respective country's regulatory requirement.