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Detection of GM maize in food by multiplex, semi-quantitative and real-time PCR with restriction enzyme reaction
|摘要:||In this study, a semi-quantitative PCR was developed to detect GM (genetically modified ) maize that contain insecticidal cryIA(b) protein from Bacillus thuringiensis. We chose two primers, 35SP-F/R (205 bp) and cryIA-4-5'/4-3' (152 bp), for detecting transgene in maize. In addition, primer of INVA/INVB (122 bp) was used to amplify the endogenous gene. The result indicated that the correlation coefficients of simplex and duplex semi-quantitative PCR reaction were 0.9638 and 0.975, respectively. The detection limit of multiplex PCR with three primers was 0.1%. In Taiwan, food products or raw material that contained more than 5% GM maize are required to put on the label. This method is sensitive enough to meet this requirement.
For distinguishing various maize events, a restriction enzyme method with PCR procedures was successfully developed. Our results showed that when three Bt maizes, including MON810, Bt11 and Event176 were amplified with the primers described as above and treated with restriction enzyme RsaI, byproducts with 117 bp+35 bp, 86 bp+66 bp and 152 bp were formed, respectively. Detection of transgenic gene in process product by our semi-quantitative PCR and real-time PCR showed high correlation of 0.9875. Result with more than 7% or less than 2% DNA content by using semi-quantitative PCR are determined to be positive (>5%) or negative (<5%), respectively. Only the results with the DNA content in the range of 2% - 7% needed to be farther confirmed with quantitative PCR method.
The semi-quantitative PCR method with restriction enzyme treatment not only provides an easy way to detect GM maize in processed food products, but also with the ability to distinguish various Bt maize.|
本研究為利用半定量聚合酶連鎖反應檢測基因改造玉米中具有來自於蘇力菌（Bacillus thuringiensis）抗蟲蛋白cryIA(b)之Bt抗蟲玉米。首先，由參考文獻收錄之引子組進行二次確認，包含偵測玉米轉殖基因的引子35SP-F/R (205 bp)與cryIA-4-5’/4-3’ (152 bp)以及擴增玉米內生性基因的引子INVA/INVB (122 bp)。結果顯示，進行單套式與雙套式PCR，其相關係數分別可達0.9638 和 0.975，且利用三組引子進行多套式PCR，其檢測靈敏度可達0.1%，這樣的結果符合我國基因改造食品之標示標準。 為區分基因改造玉米的品系，本研究利用限制酶RsaI作用於PCR產物，再利用次產物的片段大小，可將Bt玉米區分為三種玉米品系，分別為MON810: 117 bp+35 bp、Bt11: 86 bp+66 bp與不被作用的Event176: 152 bp。另外，將半定量之檢測結果與即時定量PCR比較，兩檢測系統間之相關性可達0.9875，此半定量分析方法可將基因改造玉米加工品成分區分在7％以上以及2%以下，若樣品落於2%-7%之範圍內，則可再利用即時定量方法進一步確認即可。 本研究所發展之多套式半定量PCR複合限制酶作用，不僅可易於檢測出基因改造玉米加工品中之基因改造成分，更可利用限制酶剪切來 區分不同品系之Bt玉米。
|Appears in Collections:||食品暨應用生物科技學系|
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