利用 QCPCR 進行基因改造玉米樣品檢測追蹤

Abstract

本研究由全臺各地取得 107個玉米樣品，每個玉米經三取樣共選取321個試驗樣品進行基改玉米篩選。先以 35S Promoter 與 NOS Terminator 此二基因配合玉米內源性基因 zein，以 Multiplex PCR 篩選 GMO。其中檢測出兼具 35S Promoter 與 NOS Terminator 者佔 3.12%、僅有 35S Promoter者佔 1.25%，估計總共 GMO 玉米佔 4.36%。再以Onishi 等學者於2005年所提出之八品系基改玉米專一性篩選系統進行品系檢定。所篩選出含有NK603 雜交品系之 GMO 玉米，則佔了總 GMO 玉米的 71.4%。完成市售玉米之定性檢測後，將篩選出之 GMO樣品進行定量分析。傳統 QCPCR 在應用上的缺點有二：( 1 ) QCPCR產物電泳分析，competitor 必須大於原PCR目標序列 20∼40 bp 以上，否則電泳分析便難以將二者分離。然而，引子對之競爭誤差此時變得無法避免。( 2 ) 接續電泳結果後之影像分析，常因染色與影像系統的靈敏度而影響結果。因此，本研究運用 HPLC 系統來提升 QCPCR 的檢測效益。並在內標準質體的設計上，參考 Yang 等學者於 2007 年所發表能同時應用在九種特定玉米品系之 real-time PCR 分析之標準參考質體，加以修改其基因片段長度，使其亦能做為 QCPCR 檢測之內標準質體。利用引子設計與限制

The 107 maize samples, collected from stores, retailers and supermarkets, were used for serial tests of genetic modified organism (GMO). Total 321 test samples were picked from these 107 maize samples. First test was performed by Multiplex PCR, using primers designed from 35S Promoter, NOS Terminator and zein, to identify GMO maize. Then, GMO maize was further identified for their types by using the method of recognizing eight different types of GMO maize, published in 2005 by Onishi. In the detection of different types of GMO maize, 71.4% test samples was detected to be NK603. The test samples, detected to be GMO maize, will be quantity. There are disadvantages in the past way in using QCPCR, such as that the size of PCR products between competitor and target should be large enough to be separated on agarose gel. The image took to analysis will also be affected by factors like staining time of EtBr and the system used to capture the image. Therefore, we try to overcome those problems by taking HPLC into QCPCR. A reference plasmid, applied by Yang to real-time PCR for nine specific types of maize, was modified to form a standard plasmid for the test of QC-PCR. The modified plasmid, inserted with 6 bp restriction site, specific for the use in quantitation of NK603, could be put into comparing real-time PCR and QC-PCR equally. The two peaks could be separated in 40 seconds by HPLC system in this condition: 5 mM NaCl gradient per min and 0.75 mL per min flow rate. HPLC system will further use in rapid, repetitive and accurate quantitation of GMO.