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The Development and Application of Polymerase Chain Reaction Techniques on Detection of Genetically Modified Foods-The Collaboratory Studies
|作者:||方繼||關鍵字:||應用研究;genetically modified food;生物技術, 食品科技;基因改造食品;多套式PCR;半定量PCR;聯合評估;multiplex PCR;semi-quantitative PCR;collaboratory evaluation||摘要:||
近年來由於分子生物學之進步，基因改造技術廣泛應用於微生物、植物及動物上，世界農業因生物技術之應用為世界人口提供充足糧食帶來新契機。基因改造生物（Genetically Modified Organisms, GMO）於近年來備受關注，其相關研究亦日益增多，目前已有不少基因改造作物於市場上流通。我國進口之大豆中已逾50%為基因改造大豆，其中又以Roundup ReadyTM品種為主。為有效管理GMO，行政院衛生署目前已公告「基因改造食品安全性評估方法」及「基因改造食品標示辦法」，為我國基因改造食品之管理準則。目前基因改造食品之種類日益繁多，雖有許多方法均可檢測基改食品之存在，然國際間仍缺乏一套驗證食品中是否含有基因改造生物檢測方法之制度。若能於國內建立數個比對實驗室，對於目前已發表之檢測方法進行不同實驗室間之驗證，相信對建立檢驗基因改造食品各項方法之標準化及對政府相關機構管理基因改造食品之各項事宜定有所助益。本研究室於行政院衛生署之補助下，於九十一年二月起進行多套式聚合?鏈鎖反應（Multiplex PCR）鑑定方法之開發及建立比對實驗室，評估其應用於檢驗基因改造大豆及市售產品之可行性，實驗過程為針對Roundup ReadyTM基因改造大豆之轉殖基因設計不同引子，分別為35SP-F/R的143 bp、NOS-F/R的189 bp、EPSPS-F/R的314 bp及35SP-F/CTP-R的256 bp，並利用LEC-F/R的175 bp作為品種特性基因之鑑定。結果顯示，利用上述引子35SP-F/CTP-R及LEC-F/R進行多套式PCR方法鑑定基因改造大豆及市售產品，可鑑別出含基因改造大豆之檢體。此外，為瞭解市售產品中之GMO比例，本實驗亦將市售產品之PCR產物量與不同GMO比例之DNA標準曲線作比對並進行半定量分析，以推斷市售產品中之GMO比例。本研究所開發之多套式PCR方法可容易檢出基因改造大豆之檢體，而半定量分析方法可應用於粗略區分基因改造大豆含量之限度，該方法操作簡便，適用於大量樣品之篩檢。本申請案擬以PCR技術(定性及半定量方式)建立基因改造食品檢驗方法之比對實驗室，除蒐集國內外已發表檢測基因改造食品之定量方法與本研究室所開發之半定量PCR進行比對及再驗證外，並參與衛生署藥檢局PCR檢測方法之聯合評估工作。樣品將以大豆為主軸，另外並蒐集及開發方法應用於基因改造食品加工製程之探討，以確定加工製程對檢驗方法其檢驗靈敏度及檢驗限度之影響。期藉由此檢驗方法之建立，能對基因改造食品之檢驗、定量與標示具有相當大的助益。
The development of modern molecular biology not only has broaden the application of genetic engineering techniques in microbiology, plants and animals, but also brought the new sources for the world food supplies. Genetically modified organism has received much attention in resent years and researchers have put their time and efforts in this promising area. In Taiwan, more than 50% of imported soybeans are genetically modified soybeans, which are mainly Roundup ReadyTM species. Department of Health (DOH), Taiwan, R.O.C has promulgated ？HHH？HHH？HHH？HHH？HHH？HHH？HHH？HHHGuidance of Safety Assessment for Genetically Modified Foods？HHH？HHH？HHH？HHH？HHH？HHH？HHH？HHH and ？HHH？HHH？HHH？HHH？HHH？HHH？HHH？HHHLabeling of GM Foods？HHH？HHH？HHH？HHH？HHH？HHH？HHH？HHH that forms the foundation of administration of GM foods in Taiwan.Various methods for detection of GMO have been developed by different researchers and organizations around the world; however, very few validation or collaboratory studies of these methods are available yet. Since the promulgation of these acts mentioned above, effective and correct methods to detect GMO are necessary for DOH in Taiwan. The establishment of collaboratory laboratories and validation of the detection methods for GMO will enhance the standard operation procedures among these institutes. The Applied microbiology and biotechnology laboratory (National Chung Hsing University) has started the development of multiplex PCR technique to detect the GM soybean in feed and food by the financial support of DOH (Taiwan) since 1992. Various primers were designed based on the gene sequences of Roundup ReadyTM GM soybeans. The main reference gene loci were 35SP-F/R (143 bp), NOS-F/R (189bp), EPSPS-F/R (314 bp), and 35SP-F/CTP-R), and LEC-F/R (175) was used as an identification gene for soybean specie. The results showed that multiplex PCR with primers of 35SP-F/CTP-R and LEC-F/R were effective for detection of GM soybeans. We also developed a semi-quantitative multiplex PCR technique, which based on regression and in the aid of computer software. This semi-quantitative multiplex PCR proved to be simple to used and suitable for mass screening of GMO.This proposal intends to apply the qualitative and semi-quantitative PCR techniques for establishment the collaboratory laboratory among several institutes in Taiwan. Detection methods for GMO will be collected around the world and compared to the methods that have been developed by our laboratory. Comparison and validation of the methods developed by DOH (Taiwan) and our methods will also be conducted. Soybean will be the major target for this study, the influence the environmental factors such as processing procedures on the detection limit and sensitivity of the PCR technique will be investigated as well.
|Appears in Collections:||食品暨應用生物科技學系|
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