基因改造大豆及四種品系玉米檢測方法之研究

Abstract

近年來世界各國逐漸重視基因改造食品的應用和安全性，並制定相關標準與法規予以規範與管理，我國基因改造食品於西元2003年起實行強制標示。本研究以35S-F/35-R和nos-F/nos-R兩組引子進行複式聚合酶鏈鎖反應，可正確檢測出一種基因改造大豆及四種基因改造玉米。針對基因改造玉米Bt11和Bt176之轉殖基因3’端與玉米染色體DNA相接處 (integration site) 設計具品系專一性 (event-specific) 之引子Bt11-F/Bt11-R、Bt176-F/Bt176-R；針對基因改造玉米MON810和GA21之基因改造5’端與玉米染色體DNA相接處設計具專一性之引子MON810-F/MON810-R和GA21-F/GA21-R。以即時聚合酶鏈鎖反應TaqMan進行定量，四種基因改造玉米品系Bt11、Bt176、MON810、GA21之平均內標值分別約為0.74、0.62、0.26、1.41；定量檢測系統之正確性檢驗，CV範圍介於3.69 ~ 35.81%間，偏移值介於0.08 ~ 0.24%間。針對基因改造大豆RRS之轉殖基因3’端與大豆染色體DNA相接處設計具有專一性之引子RRS-F/RRS-R；以即時聚合酶鏈鎖反應SYBR Green I dye進行定量，基因改造大豆RRS之平均內標值約為1.15。大量樣品取樣方法，設計出三種類型20種取樣方法：類型Ⅰ比較不同樣本大小；類型Ⅱ比較階段式取樣之差異；類型Ⅲ比較複合樣品間之差異，類型Ⅲ取樣方法17複合樣品以三點取樣，對於原始族群也較具有代表性，檢驗上也較省時低成本，此方法可作為日後大量檢體取樣方法之參考。

The detection of GM-food has its necessity due to food safety regulation, and the labeling of GM-food has been practiced since 2003 in Taiwan. The use of multiplex PCR containing 35S-F/35S-R and nos-F/nos-R is found applicable to detect four lines of genetically modified maize: Bt11, Bt176, MON810, GA21 maize, and one line of genetically modified soybean: Roundup Ready soybean. We further designed the event-specific primers to target the 3’ integration sites of Bt11 and Bt176 and the 5’ integration sites of MON810 and GA21. The quantification of Bt11, Bt176, MON810 and GA21 were carried out by using Lightcycler Instrument with TaqMan Kit. The coefficient values of Bt11, Bt176, MON810 and GA21 were 0.74, 0.62, 0.26 and 1.41. The accuracy of real-time Q-PCR detection methods, expressed as coefficient of variation for Bt11, Bt176, MON810 and GA21 varied from 3.69% to 35.81% and bias range from 0.08% to 0.24%. We designed the event-specific primers to target the 3’ integration sites of RRS. We developed the event-specific real-time detection and quantification of RRS using Lightcycler Instrument with SYBR Green I dye. The coefficient value of RRS was 1.15. In the development of bulk sampling methods, we designed twenty sampling methods in three types. In type Ⅰ, different sample sizes were compared; in type Ⅱ, we compare the process of multi-stage sampling; in type III, the mixed sampling methods were studied. The results showed that the sampling method 17 in type Ⅲ could decrease variations between individuals and save more time and cost. This method is found potential to be applied in bulk sampling process in future.