유전자변형 면화 MON757, MON88702, COT67B, GHB811의 동시검출법 개발
Development of simultaneous detection method for living modified cotton varieties MON757, MON88702, COT67B, and GHB811
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초록
면화는 중요한 섬유 작물로 종자는 가축의 사료로 사용된다. 작물 생명공학은 농업 분야에서 농업적 형질과 질을 향상시키기 위해 활용되어져 왔다. 국내 식품, 사료, 가공 제품에 유전자변형 (LM) 면화의 사용이 증가함에 따라 환경으로의 LM 면화의 비의도적 유출 또한 증가하고 있다. LMO 모니터링 사업에서 수집된 LM 면화를 검정하기 위하여 국내 수입 승인된 LM 면화의 검출법 개발이 필요하다. 본 연구에서는 LM 면화 MON757, MON88792, COT67B, GHB811 4종을 대상으로 동시검출법을 개발하였다. 이벤트에 대한 유전 정보는 유럽 JRC와 농림축산검역본부에서 확보하였다. LM 면화의 동시검출법 개발을 위해 이벤트 특이적인 프라이머를 설계하였으며 특이적인 증폭을 확인하였다. 특이도 검정, 무작위 표준물질 혼합물 분석, 검출한계 분석을 통하여 동시검출법의 정확도와 특이도를 검증하였다. 그 결과 본 동시검출법은 각각의 이벤트를 검출할 수 있으며 LM 표준물질을 활용하여 특이도를 검정하였다. 또한 무작위 표준물질 조합도 정확하게 검출할 수 있다. 검출한계 분석에서는 25 ng의 미량의 주형 DNA로 단회 분석으로 검출이 가능하다. 결론적으로 4종의 LM 면화 동시검출법을 개발하였으며 LM 면화 자생체 분석에 활용될 것으로 사료된다.
Abstract
Cotton is an important fiber crop, and its seeds are used as feed for dairy cattle. Crop biotechnology has been used to improve agronomic traits and quality in the agricultural industry. The frequent unintentional release of LM cotton into the environment in South Korea is attributed to the increased application of living modified (LM) cotton in food, feed, and processing industries. To identify and monitor the LM cotton, a method for detecting the approved LM cotton in South Korea is required. In this study, we developed a method for the simultaneous detection of four LM cotton varieties, MON757, MON88702, COT67B, and GHB811. The genetic information of each LM event was obtained from the European Commission-Joint Research Centre and Animal and Plant Quarantine Agency. We designed event-specific primers to develop a multiplex PCR method for LM cotton and confirmed the specific amplification. Using specificity assay, random reference material (RM) mixture analysis and limit of detection (LOD), we verified the accuracy and specificity of the multiplex PCR method. Our results demonstrate that the method enabled the detection of each event and validation of the specificity using other LM RMs. The efficiency of multiplex PCR was further verified using a random RM mixture. Based on the LOD, the method identified 25 ng of template DNA in a single reaction. In summary, we developed a multiplex PCR method for simultaneous detection of four LM cotton varieties, for possible application in LM volunteer analysis.
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