五種轉殖品項基因改造玉米（Bt11、Event176、GA21、MON810及T25）即時定量PCR檢驗方法之建立   全文下載

Establishment of Quantitative Method for Five Events of Genetically Modified Maize (Bt11, Event176, GA21, MON810 and T25) by Real-Time QPCR

為配合基因改造食品標示制度之施行，本研究著重於即時定量聚合?鏈反應（real-time quantitative polymerase chain reaction, real-time QPCR）方法之開發，以定量檢測基因改造玉米。針對五種轉殖品項Event1 76 及Bt11 （Syngenta公司）、MON810 及GA21 （Monsanto公司）及T25 （Aventis公司）之殖入基因設計產品特異性引子及探針，並進行real-time QPCR測試。同時，針對每種轉殖品項基因改造玉米構築一含轉殖基因及內部對照基因標的之質體，以作為定量參考分子，並進一步使用構築質體及測試檢體測試，以評估本定量系統。結果顯示，以構築之質體經由系列稀釋所製作之標準曲線， 其獲得之slope值介於-3.31至-3.45，correlation介於0.99至1.0。以本研究發展之方法定量測試含1%、2%或5%基因改造玉米之測試檢體，1%測試檢體之平均值（mean）介於1.01%至1.23%，2%測試檢體介於2.00%至2.31%，5%測試檢體介於4.46%至5.55%，標準偏差（standard deviation, S.D.）介於0.05至0.46，變異係數（coefficient of variance. C.V.）介於5%至15%。本方法之定量極限（lintits of quantitation, LOQ）為0.1% （w／w）。除此，以本研究發展之方法測試精準度試驗檢體，可獲得優異結果。"

In order to meet the implementated labeling requirement, this study focused on the development of real-time quantitative polymerase chain reaction (real-time QPCR) method to detect genetically modified maize (GM-maize). Primers and probes specific for inserted genes in Event176, Bt11 (Syngenta company), MON810, GA21 (Monsanto company) and T25 GM-maize (Aventis company) were designed and used to conduct the real-time QPCR assays. A plasmid containing both transgene and internal control gene targets on the same molecules for each GM-maize event was also constructed as quantitative reference molecules. Further, constructed plasmids and test samples were applied to validate this quantitative system. Results showed that the slope of the standard curve generated by serial dilution of constructed plasmids was in the range of -3.31~-3.45 with a correlation of 0.99~1.0. Test samples spiked 1%, 2% and 5% GM-maize were quantitated by the method developed in the study. The mean values of 1%, 2% and 5% test sample were between 1.01%~1.23%, 2.00%~2.31% and 4.46%~5.55%, respectively. The standard deviation (S.D.) ranged from 0.05 to 0.46 with coefficient variance (C.V.) of 5%~15%. The limit of quantitation was 0.1% (w/w) for these GM-maize crops. In addition, proficiency test samples were analyzed by the method developed in this study and good results were obtained. 為配合基因改造食品標示制度之施行，本研究著重於即時定量聚合酶鏈反應（real-time quantitative polymerase chain reaction, real-time QPCR）方法之開發，以定量檢測基因改造玉米。針對五種轉殖品項Event1 76 及Bt11 （Syngenta公司）、MON810 及GA21 （Monsanto公司）及T25 （Aventis公司）之殖入基因設計產品特異性引子及探針，並進行real-time QPCR測試。同時，針對每種轉殖品項基因改造玉米構築一含轉殖基因及內部對照基因標的之質體，以作為定量參考分子，並進一步使用構築質體及測試檢體測試，以評估本定量系統。結果顯示，以構築之質體經由系列稀釋所製作之標準曲線， 其獲得之slope值介於-3.31至-3.45，correlation介於0.99至1.0。以本研究發展之方法定量測試含1%、2%或5%基因改造玉米之測試檢體，1%測試檢體之平均值（mean）介於1.01%至1.23%，2%測試檢體介於2.00%至2.31%，5%測試檢體介於4.46%至5.55%，標準偏差（standard deviation, S.D.）介於0.05至0.46，變異係數（coefficient of variance. C.V.）介於5%至15%。本方法之定量極限（lintits of quantitation, LOQ）為0.1% （w／w）。除此，以本研究發展之方法測試精準度試驗檢體，可獲得優異結果。