利用質體參考物質以及多重聚合酶連鎖反應建構基改馬鈴薯之檢測與定量方法

杜元凱、馮昱維、吳明哲、王熙宇、陳述、陳涵葳。2016。利用質體參考物質以及多重聚合酶連鎖反應建構基改馬鈴薯之檢測與定量方法。台灣農業研究 65(1):18-30。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。

關鍵字

基因改造；馬鈴薯；即時聚合酶鏈鎖反應；質體參考物質

並列摘要

Tu, Y. K., Y. W. Feng, M. T. Wu, S. Y. Wang, S. Chen, and H. W. Chen. 2016. Development of detection and quantification methods for transgenic potato using multiplex PCR and plasmidic reference material. J. Taiwan Agric. Res. 65(1):18-30. 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%.

並列關鍵字

Genetically modified ； Potato, Real-time PCR ； Plasmidic reference material

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利用質體參考物質以及多重聚合酶連鎖反應建構基改馬鈴薯之檢測與定量方法

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