Development and Validation of a Multiplex PCR Assay for the Simultaneous Detection of Four LM Maize Events in Environmental Monitoring
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초록
This study focuses on developing a multiplex PCR assay for the simultaneous detection of four specific living modified (LM) maize varieties (DP-004114-3, MON89034, MON88017, and MON87403) approved for use in South Korea. The increasing use of LMO maize in food, feed, and industrial applications has heightened concerns about unintentional environmental releases, underscoring the need for reliable detection methods. Genetic information for each LMO event was obtained, and event-specific primers were designed to ensure accurate amplification. The developed multiplex PCR method underwent rigorous validation through specificity assays, analysis of certified reference material (CRM) mixtures, and limit of detection (LOD) testing. The assay effectively detected all target events with high sensitivity, capable of identifying as little as 12.5 ng μL–1 of genomic DNA. Furthermore, this method was utilized with suspicious samples gathered during the monitoring of unintentional LMO releases in natural settings, confirming its utility in practical applications. Our results show that the multiplex PCR assay is a robust and efficient method for the detection and monitoring of LM maize in both environmental and regulatory contexts. This technique holds considerable potential to safeguard natural ecosystems by preventing unintentional LMO releases and ensuring adherence to biosafety regulations.
Abstract
This study focuses on developing a multiplex PCR assay for the simultaneous detection of four specific living modified (LM) maize varieties (DP-004114-3, MON89034, MON88017, and MON87403) approved for use in South Korea. The increasing use of LMO maize in food, feed, and industrial applications has heightened concerns about unintentional environmental releases, underscoring the need for reliable detection methods. Genetic information for each LMO event was obtained, and event-specific primers were designed to ensure accurate amplification. The developed multiplex PCR method underwent rigorous validation through specificity assays, analysis of certified reference material (CRM) mixtures, and limit of detection (LOD) testing. The assay effectively detected all target events with high sensitivity, capable of identifying as little as 12.5 ng μL–1 of genomic DNA. Furthermore, this method was utilized with suspicious samples gathered during the monitoring of unintentional LMO releases in natural settings, confirming its utility in practical applications. Our results show that the multiplex PCR assay is a robust and efficient method for the detection and monitoring of LM maize in both environmental and regulatory contexts. This technique holds considerable potential to safeguard natural ecosystems by preventing unintentional LMO releases and ensuring adherence to biosafety regulations.
참고문헌 (16)
- Adugnaw, A., "The role of modern biotechnology in the fight against the current and the future climate change", Journal of Advances in Biology & Biotechnology, 27(5), pp.595~610 (2024)[DOI]
- Brownie, J., "The elimination of primer-dimer accumulation in PCR", Nucleic Acid Research, 25(16), pp.3235~3241 (1997)[DOI]
- Choi, W., "A multiplex PCR method for the detection of genetically modified alfalfa(Medicago sativa L. )and analysis of feral alfalfa in South Korea", Proceedings of the National Institute of Ecology of the Republic of Korea, 1, pp.83~89 (2020)
- Eum, S. -J., "Event specific multiplex PCR method for four genetically modified cotton varieties, and its application", Journal of Applied Biological Chemistry, 62, pp.52~ (2019)[DOI]
- ISAAA, "Global status of commercialized Biotech", GM crops in 2019: Biotech crops drive socio-economic development and sustainable environment in the new frontier, 2019
- James, C., "Global review of the field testing and commercialization of transgenic plants, 1986 to 1995: The first decade of crop biotechnology. International Service for the Acquisition of Agri-biotech Applications", Briefs No. 1. ISAAA, 31~ (1995)
- Jo, B. -H., "Multiplex PCR method for environmental monitoring of approved LM cotton events in Korea", Journal of Plant Biotechnology, 43, pp.91~98 (2016)[DOI]
- "KBCH Trend Report" (2021)[URL]
- "KBCH Trend Report" (2023)[URL]
- Kim, D. W., "Development of a multiplex PCR assay to monitor living modified cottons in South Korea", Journal of Applied Sciences, 9, pp.2688~ (2019)[DOI]
- Kim, I. R., "Monitoring living modified canola using an efficient multiplex PCR assay in natural environments in South Korea", Journal of Applied Sciences, 10, pp.7721~ (2020)[DOI]
- Kim, I. R., "Development of simultaneous detection method for living modified cotton varieties MON757, MON88702, COT67B, and GHB811", Korean Journal of Environmental Biology, 39(4), pp.415~422 (2021)[DOI]
- Markoulatos, P., "Multiplex polymerase chain reaction: A practical approach", Journal of Clinical Laboratory (2002)[DOI]
- National Institute of Ecology. Study on Environmental Monitoring and Post-management of LMO, "NIE", National Institute of Ecology, ~ (2023)
- Park, J. H., "Development of a multiplex PCR method for identification of four genetically modified maize lines and its application in living modified organism identification", Journal of Plant Biotechnology, 47, pp.309~315 (2020)[DOI]
- Shin, S. Y., "Four multiplex PCR sets of 11 LM Maize for LMO environmental monitoring in Korea", Journal of Plant Biotechnology, 43, pp.473~478 (2016)[DOI]