以PCR方法檢測基因改良大豆及玉米   全文下載

Detection of Genetically Modified Soybeans and Maize by the Polymerase Chain Reaction Method

本研究以PCR 方法探討鑑別檢測基因改良大豆與基因改良玉米之可行性。針對Roundup Ready（Monsanto 公司）GM ──大豆及Event 176（Novartis／Ciba-Geigy 公司）GM──玉米產品插入基因與品種特性基因選定不同引子，進行PCR 方法檢測。用以鑑別GM ──大豆之引子共四對，分別為35S（35S-promoter, 源自cauliflower mosaic virus）、NOS（nopaline synthase-terminator,源自Agrobacterium tumefaciens）、EPSPS（5-enolpyruvylshikimate-3-phosphate synthase，源自A. tumefaciens strain CP4）及LE（品種特性基因lectin）。而鑑別GM ──玉米之引子則採用三對，分別為CDPK-cry （pollen-specific calciumdependentprotein kinase promoter -delta-endotoxin ，分別源自玉米及Bacillus thuringiensis subsp. kurstaki）、cryIA（b）（delta-endotoxin，源自B. thuringiensis subsp. kurstaki）及ivr（品種特性基因invertase）。結果顯示，大豆檢體以35S 及EPSPS 引子檢測時，其最低檢測量均為0.1%（w／w），NOS 則為1%（w／w）；檢體並以LE 引子── PCR 反應確定均為大豆產品。至於玉米檢體之測試結果，使用CDPKcry引子其最低檢測量為0.1%（w／w）， cryIA（b）為2%（w／w），並經由Invertase 引子── PCR 反應確定為玉米產品。此外，GM──大豆之35S-PCR產物，進一步以限制酵素Xmn I進行切割確認， 195 bp產物切成80 bp與115 bp產物，而NOS之180 bp 產物則以限制酵素Nsi I切為84 bp 與96 bp，確認PCR產物。結果顯示本報告所使用之PCR方法能區分一般與基因改良之大豆及玉米。"

The feasibility of detecting genetically modified (GM) soybeans and GM maize by a polymerase chain reaction (PCR) method is determined. Primers specific for inserted genes and crop endogenous genes in Roundup Ready soybeans (Monsanto company) and Event 176 GM maize (Novartis/Ciba-Geigy company) were applied. Four pairs of primers, namely, 35S (35S-promoter, originated from cauliflower mosaic virus), NOS (nopaline synthase-terminator, derived from Agrobacterium tumefaciens), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, obtained from A. tumefaciens strain CP4) and LE (endogenous gene lectin) were used to identify the GM soybeans. An additional three pairs of primers, including CDPK-cry (pollen-specific calcium-dependent protein kinase promoter - delta-endotoxin, acquired from maize and Bacillus thuringiensis subsp. kurstaki, respectively), cryIA(b) (delta-endotoxin, evolved from B. thuringiensis subsp. kurstaki) and ivr (endogenous gene invertase) were directed to confirm the GM maize. Using 35S and EPSPS as primers, the method showed a limit of detection for samples containing 0.1% (w/w) of GM soybeans and containing 1% (w/w) of GM soybeans when NOS primers were applied. All soybean samples were evidenced by LE primer-PCR as soybean products. Detection limits of 0.1% (w/w) of GM maize in raw material using CDPK-cry primers and 2% (w/w) of GM maize with cryIA(b) were established. The maize products were also approved by Invertase primer-PCR. To further confirm detection of the target GM soybean by PCR, the 195 bp fragment, amplified from 35S-PCR, digested with endonuclease XmnI resulted in 80 and 115 bp fragments, while digesting the 180 bp amplified products from NOS-PCR using endonuclease NsiI yielded 96 and 84 bp fragments. The data further revealed that the PCR method can sufficiently differentiate GM soybeans and maize from non-GM products. 本研究以PCR 方法探討鑑別檢測基因改良大豆與基因改良玉米之可行性。針對Roundup Ready（Monsanto 公司）GM ──大豆及Event 176（Novartis／Ciba-Geigy 公司）GM──玉米產品插入基因與品種特性基因選定不同引子，進行PCR 方法檢測。用以鑑別GM ──大豆之引子共四對，分別為35S（35S-promoter, 源自cauliflower mosaic virus）、NOS（nopaline synthase-terminator,源自Agrobacterium tumefaciens）、EPSPS（5-enolpyruvylshikimate-3-phosphate synthase，源自A. tumefaciens strain CP4）及LE（品種特性基因lectin）。而鑑別GM ──玉米之引子則採用三對，分別為CDPK-cry （pollen-specific calciumdependentprotein kinase promoter -delta-endotoxin ，分別源自玉米及Bacillus thuringiensis subsp. kurstaki）、cryIA（b）（delta-endotoxin，源自B. thuringiensis subsp. kurstaki）及ivr（品種特性基因invertase）。結果顯示，大豆檢體以35S 及EPSPS 引子檢測時，其最低檢測量均為0.1%（w／w），NOS 則為1%（w／w）；檢體並以LE 引子── PCR 反應確定均為大豆產品。至於玉米檢體之測試結果，使用CDPKcry引子其最低檢測量為0.1%（w／w）， cryIA（b）為2%（w／w），並經由Invertase 引子── PCR 反應確定為玉米產品。此外，GM──大豆之35S-PCR產物，進一步以限制酵素Xmn I進行切割確認， 195 bp產物切成80 bp與115 bp產物，而NOS之180 bp 產物則以限制酵素Nsi I切為84 bp 與96 bp，確認PCR產物。結果顯示本報告所使用之PCR方法能區分一般與基因改良之大豆及玉米。