研發定量分析系統以檢定基因改造玉米

Abstract

本研究目的在於建立能檢定基因改造玉米混雜率的定量系統，以聚合酵素連鎖反應為基礎技術的即時聚合酵素反應，針對Bt11、176、GA21、NK603、MON810、MON863、TC1507等七個基因改造玉米品項設計具有專一性的定量系統。首先以定序取得七個基因改造玉米品項的部份轉殖基因序列，進而針對其中具有專一性的片段來作為設計引子對及探針，並將所挑選的專一性片段加上玉米內生基因片段zSSIIb、轉殖基因通用啟動子P35S與終結子Tnos接合轉殖進質體當作定量用途的標準物質，最後實際以認證標準物質 (certified reference material, CRM) 驗證本研究中建立的定量系統定量的精準度。認證標準樣品包含各品項混雜比例從0~10.0%不等，經過驗證後此定量系統的偏估值變動的分布主要在 %以內而變異係數變動範圍主要在20%以內，顯示此定量系統可得到可靠的定量結果。

The purpose of this study is to establish a quantification system based on real-time PCR for 7 most common genetically modified events of maize. Targeted events include Bt11, 176, GA21, NK603, MON810, MON863 and TC1507. Construct specific primers and probes were designed according to the partial sequences of inserts verified by direct sequencing of PCR amplicons of primers found in the literatures. Specific amplicons of the above mentioned events and maize endogenous DNA sequence zSSIIb, cauliflower mosaic virus 35S promoter, nopaline synthase terminator were appended and inserted into PGEM-T vector. A PstI digested linear form of transformed vector was quantified and used to establish the standard curves of each event. Certified reference materials ranging from 0 to 10% were used to validate the accuracy and precision of this quantification system. In most of the cases, the bias ranged from -30% to 30% and coefficients of variation were less then 20%. These results have proved that the GMO quantification system developed in this study is reliable in practice.