(65(1):18-30)Development of Detection and Quantification Methods for Transgenic Potato Using Multiplex PCR and Plasmidic Reference Material

基改作物一般無法以外觀型態加以區分，而透過以聚合酶連鎖反應 (polymerase chain reaction; PCR) 所發展的各項檢測技術，則可明確進行篩選與區別。本研究針對基因改造馬鈴薯EH92-527-1 品系，利用該品系邊界序列 (flanking sequence) 與馬鈴薯UGPase 序列將其構築於質體參考物質pGEM-EH92，利用即時聚合酶鏈鎖反應 (real-time PCR) 建立檢量線：決定係數R2 = 0.99，檢量範圍最低為20 個拷貝數，3% (w/w) 偏誤值(bias) 為16.3%，相對標準誤差 (relative standard error) 介於6–12.9%。顯示其確實可實質應用於檢測基改馬鈴薯EH92-527-1 品系，此質體參考物質兼具容易保存、便宜與穩定佳等優點。另外，以EH92-527-1 品系之RNApol (RNA polymerase) 與nptII (Neomycin phosphotransferase II)、反義GBSS (granule bound starch synthase)基因為目標，建構多重聚合酶鏈鎖反應 (multiplex PCR) 反應，可得最低檢測極限為3%，由此可大幅縮短檢測時間、檢測成本，並提升檢測準確度。 The objectives of this research were to establish a new quantification method and a fast multitarget PCR method for genetically modified potato event, EH92-527-1. A plasmidic reference material (PRM), pGEM-EH92, was constructed and used as a calibrant to quantify content of EH92-527-1. Results showed the lowest of quantification was 0.1% (w/w), bias and relative standard error were 16.3% and 6–12.9%, respectively, at 3% (w/w) true value. It indicated that the quantification method by means of PRM was able to practically determine the EH92-527-1 event content of mixed sample above 3%. When RNApol (RNA polymerase), nptII (Neomycin phosphotransferase II) and antisense GBSS (Granule bound starch synthase) sequences of EH92-527-1 event were used as multiplex PCR target, the lowest of qualification of multiplex PCR method was 3%