發展多套式和定量PCR方法檢測基因改造大豆及市售產品   全文下載

Development of Multiplex and Quantitative PCR Assay to Detect Genetically Modified Roundup Ready Soybean in Foods

本研究為利用多套式聚合瓷鏈反應（Multiplex PCR）鑑定方法，評估鑑別檢測基因改造大豆及其市售產品之可行性。實驗過程針對Roundup Ready（Monsanto 公司，美國）基因改造大豆之轉殖基因設計四對引子，分別為35sP（Cauliflower mosaic virus 35S promoter）、nosT（Agrobacterium tumefaciens nopaline synthase terminator）、35sP/CTP（Petunia hybrida EPSPS chloroplast transit peptide），並利用Lec（Lectin）作為品種特性基因之鑑定。以上述引子35sP/CTP及品種特性基因Lec進行多套式PCR方法鑑定基因改造大豆及其市售產品之結果顯示，大豆檢體以35sP及35sP/CTP引子檢測時，其最低檢測量均為0.01%（w/w），nosT及多套式PCR引子（35sP/CTP和Lec）則為0.1%（w/w）。PCR產物並以基因改造大豆之轉殖基因進行定序確認之。以本研究所開發之多套式PCR檢測含大豆之市售產品共21件，其中有14件被檢出為基因改造大豆製品。另外，利用SYBR Green Ⅰ定量PCR定量含量為20%、10%、5%、1%（w/w）之GM-soya樣品及5%之GM-soya標準品，並進一步作迴歸分析，所得R2值為0.9683。本研究所使用之多套式PCR方法除易檢驗出基因改造大豆外，並可大幅降低檢測時間及成本，有助於基因改造食品之檢驗及管理。

The objective of this study was to develop a qualitative detection method for genetically modified (GM) soybeans using the multiplex PCR technique. Potential applications for using the developed detection method to analyze GM material in soya and its products were also evaluated. Four primers for the detection of transferred genes in Roundup Ready soybean artificially synthesized for this study included 35sP (Cauliflower mosaic virus 35S promoter), nosT (Agrobacterium tumefaciens nopaline synthase terminator), and 35sP/CTP (Petunia hybrida EPSPS chloroplast transit peptide). In addition, Lec (Lectin) primers were used to detect soybean species specificity. The results showed using either 35sP or 35sP/CTP as a primer obtained a detection limit of 0.01% (w/w) and using either primer nosT or multiplex PCR with 35sP/CTP obtained a detection limit of 0.1% (w/w). Furthermore, the transferred genes in Roundup Ready soybeans were confirmed through the isolation and sequencing of their genes. By using the multiplex PCR method developed in this study, we detected GM soybean material in 14 of the 21 soybean products obtained from the open market for this study. In addition, samples with 20%, 10%, 5% and 1% GM-soya and 5% GM-soya standard were quantitatively analyzed with SYBR Green I, with an R2 of 0.9683 when regression analysis was applied. Our results showed that, in addition to readily detecting GM soybean material, the multiplex PCR method reduced detection time and costs. The multiplex PCR method proposed in this paper may offer a useful tool to detect and monitor GM foods.