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

Abstract

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 55°C and 25 cycles at 60°C) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at 60°C) 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.

Event-specific multiplex PCR method for four genetically modified cotton varieties, and its application

“…For high-sensitive detection, large amount of DNA as template is used; however, environmental sample of LMO cannot ensure DNA quality and quantity. Several testing methods released by developers or regulators and several previously reported GMO detection methods recommend that the minimum amount of purified DNA required is 40-100 ng in total reaction volume [6,10,11]. In this study, we detected LMOs using the newly developed multiplex PCR method, which required only 12.5 ng of purified DNA.…”

“…Among the multiplex detection methods for major GM crops, those for cotton have not been well studied because cotton is not a food crop. Multiplex PCR methods have been developed for MON15985, MON531, GHB614, MON88913, LLCOTTON25, and MON1445-2 [10], GHB119 and 281/3006 [17], and GHB119 and T304-40 [3]. In this study, a conventional multiplex PCR method for COT102, MON88701, T304-40, and DAS-81910-7 was newly developed to identify approved GM cotton varieties in Korea.…”

“…The multiplex PCR results with randomly mixed RM mixtures suggested that this method can identify all LMO stacks, including the four LM cotton varieties that will be approved in the future. The multiplex PCR method was applied to identify LM samples, which were collected from environmental monitoring [9][10][11]. To verify the application of multiplex PCR method in LMO monitoring, we used LM cotton samples collected from an LMO monitoring project in the Republic of Korea in 2018.…”

Development of a Multiplex PCR Assay to Monitor Living Modified Cottons in South Korea

“…Since 2009, the MOE and National Institute of Ecology (NIE) have been conducting The Environmental LMO Monitoring and Post-Management Project [9,10]. In the project, the natural environmental leakage of LM crops such as cotton, maize, soy bean, and canola was monitored and the risk assessment and management of released LMOs was performed [11][12][13]. A specific LMO detection method is crucial to determine whether the samples collected in this project contain LMOs [14,15].…”

“…Recently, a total of 30 LM cotton events including single and stack LMOs were approved in South Korea. A previous study reported LM cotton multiplex detection methods for six events [13]. As approval and unintentional release of LM cotton events have been increasing every year, we needed to develop a new method which can detect almost approved LM cotton events.…”