Multiplex PCR method for environmental monitoring of approved LM cotton events in Korea

The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at and 25 cycles at ) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at ) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.