Event-specific loop-mediated isothermal amplification for living modified cotton MON88701, MON531, MON15985, MON88913, and COT102

Abstract

AbstractGossypium hirsutum L., commonly known as upland cotton, is cultivated globally for natural fiber, feed, and seed oil. To enhance agricultural productivity and quality, living modified (LM) cotton has been developed and utilized since the late 1990s. Due to environmental concerns, such as biodiversity risks associated with living modified organisms (LMOs), the Korean government, including the Ministry of Environment, has been conducting LMO natural environment monitoring and post-management projects. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect five specific LM cotton events (MON88701, MON531, MON15985, MON88913, and COT102), which were the most abundant volunteers observed from 2019 to 2021 in South Korea. The event-specific LAMP assays for the five LM cotton events were established with a 40-min reaction time using LAMP reaction buffer, Bst DNA polymerase, and event-specific primers. The limit of detection ranged from 0.01 to 1 ng/μL. The assays' specificity and sensitivity were validated through colorimetric changes, fluorescence intensity measurements, and conventional PCR. To demonstrate the practical application of the LAMP assays, we tested 22 LM cotton volunteers collected from the natural environment in 2021. Each LAMP assay event-specifically amplified the respective LM cotton volunteers. These results indicate that the developed LAMP assays are effective tools for the efficient management and detection of LM cotton in field surveys, supporting regulatory compliance and environmental monitoring efforts.