以聚合酶鏈反應法檢測大豆中基因改造成分之研究

目前已有多種基因轉殖作物開始商業化大規模生產，引起世界對於食品安全、生態影響、倫理衝擊與國際貿易等議題的重視。食用安全性是發展植物相關生物技術最關鍵性問題，目前各國對於基因轉殖生物 (genetically modified organisms, GMOs) 及基因改造食品從研究發展到產品上市過程中的管理問題，都希望能找出一個可以兼顧科技發展與民眾消費安全性的管理措施。於是基因改造食品之標示已成為全球趨勢，而完備之檢驗方法為進行標示管理之基礎。因此，如何偵測食品中是否含有GMO就成為一個重要的研究課題。故本研究針對目前世界上產量最多之基因改造大豆 (RoundupTM Ready soybean, RRS) 進行定性與定量的檢驗研究。本研究共分為兩部分，第一部分：建立一巢式聚合酶鏈反應法 (nested polymerase chain reaction, nested PCR) 檢驗食品中基因改造大豆之成份。在本研究中，巢式聚合酶鏈反應法偵測極限可達 0.0025 ng，相較於一般聚合酶鏈反應法敏感度提升了200至1000倍。對於高度加工食品如本研究所用之材料豆腐乳，因加工過程中熱、酵素與化學處理使得 DNA 嚴重降解，使用巢式聚合酶鏈反應可有效放大 DNA 抽取液存在的模版數，提高檢驗之專一性與靈敏度。一般豆腐乳之熟成需一到六個月，利用巢式聚合酶鏈反應對熟成期一到六個月100% RRS的豆腐乳均可以檢測出其基因改造成分。第二部分：以ABI PRISMTM 7700 Sequence Detection System及 light upon extension (LUXTM) primer進行即時聚合酶鏈反應法 (real-time polymerase chain reaction, real-time PCR) 檢測並定量RRS。針對RRS之轉殖基因 5’ 端與大豆染色體DNA相接處 (integration site) 的序列設計專一性引子對。以50 ng DNA進行檢測時，偵測極限可達0.05 ng (約20.5 copies)，相當於0.1%。定量方面先構築一帶有大豆內源性基因lectin部分序列與 RRS 5’端插入點附近序列之質體作為定量用之標準參考物質，進而探討此法之定量範圍，根據標準曲線之範圍推測，每一反應以200 ng DNA 進行檢測，檢測範圍為0.1% 至100% 之間，變異係數在4.30% ~ 31.22% 之間，偏移值介於-16% ~ 8% 之間。其檢測靈敏度與定量範圍皆符合歐盟及我國對基因改造食品標示標準之要求。

關鍵字

基因改造；鏈反應；巢式聚合酶豆腐乳；檢測；即時聚合；基因改造生物

並列摘要

While the transgenic plants are grown and sold widely in the global market, people pay more and more attention to the events of impacts to the environmental effects, ethical issues, global trade and food safety. For the food safety being the most momentous, every country hopes to build up a set of managements, combined the biotechnological development and consumers’ right, to restrict the technology or sale of genetically modified organisms (GMOs) or GM-food products. The development and application of reliable detection and quantitative analytical methods become essential to the implementation of labeling rules. Therefore, we performed both of qualitative and quantitative studies on the detection of GM-soybean (RoundupTM Ready soybean, RRS) which covers the most output in the world. There are two main parts in this study. In the first part, a nested PCR method has been developed to distinguish traditional soybean from transgenic Roundup Ready soybeans (RRS). Compared with the standard PCR method, the nested PCR method increased the sensitivity to additional 200 to 1000-fold. One copy of RRS genomic DNA could be detected by the nested PCR. Sufu, a traditional fermented soybean curd as a very important Chinese condiment, is a highly processed food. We used the nested PCR to detect the transgenic component in sufu every month during the whole six-month aging period. The nested PCR method showed positive results for 100% RRS made sufu in all the seven detected times from 0th day to 180th day. This study described the development and application of a sensitive and specific detection method, a nested PCR system, to detect sufu made from RRS. In the second part, we developed the event-specific real-time detection and quantification of RRS using ABI PRISMTM 7700 Sequence Detection System with light upon extension (LUXTM) primer. We designed the event-specific primers to target the junction between the 5’ integration site of RRS and endogenous gene lectin and then constructed plasmid which carried both of the targeted sequences for quantitative analysis. The detection limit of LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA which was equaled to 20.5 copies; the range of quantification was from 0.1% to 100%. The sensitivity and range of quantification successfully met the bill of the requirement of the labeling rules in European Union (EU) and Taiwan.