基因改造作物泛用複合式聚合酶鏈反應篩檢系統之建立

Abstract

本研究建立的複合式聚合酶鏈反應偵測系統，以六種轉基因元件做為放大目標。分別為 cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, neomycin phosphotransferase II (npt2) gene, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 epsps) gene 與 phosphinothricin N-acetyltransferase (pat) gene。此六種放大目標為基改作物最常使用的轉基因元件，可泛用性的篩選出大多數的基因改造作物品系。依據 multiplex PCR 產物的組合以統計軟體（SPSS 12.0）進行已商業化之基改作物品系模擬分群，相同產物組合的品系分入同一群，共得到24 組 positive groups 及一組 negative group。模擬分群結果品系數量最多的positive group 僅含有 12 個品系，因此辨認一未知樣品品系最多僅需要 12+1 次PCR 反應。且此系統所使用的引子對經驗證對放大目標具有專一性，且各組引子對之間具有相容性。

The PCR targets of this multiplex PCR detection system include six transgenic elements: cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, neomycin phosphotransferase II (npt2) gene, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 epsps) gene and phosphinothricin N-acetyltransferase (pat) gene. It can screen out most of the commercialized GM events. To calculate the classifying efficiency, we use the statistical software (SPSS ver. 12.0) to simulate the classification of all GM events according to the expected patterns of the multiplex PCR results. The classification exhibits that the biggest group of the 24 positive groups only contains 12 GM events. Therefore to identify the exact event of unknown sample by this system combined with event-specific PCR detection methods, only have to run 12+1 polymerase chain reactions. Moreover, the primer pairs of this system proved to have specificity to their targets and coamplify every target in one PCR reaction.