Taq Man探针检测转基因大豆含量方法的建立及应用

Establishment and application of TaqMan PCR for detection of genetically modified soybean

目的建立TaqMan探针法检测转基因大豆含量的实时定量PCR方法并应用于市售转基因食品含量的检测。方法以0、0.1%、0.5%、2.0%、5.0%的转基因大豆RoundUp ReadyTM为标准品(Fluka公司),以特异TaqMan探针序列:5′(FAM)-cccactatccttcgcaagaccct-3′(TAMRA),引物序列F:5′-tgatgtgatatctccactgacg-3′,R:5′-tgtatcccttgagccatgttgt-3′,用实时荧光定量PCR方法扩增转基因大豆外源基因CP4EPSPS基因,在实验室建立定量转基因大豆检测的标准曲线,并对市场抽检的豆奶粉、素鸡、热狗肠、黄豆等12种豆类食品及制品进行含量分析。结果在实验室建立了TaqMan探针实时荧光定量PCR检测转基因大豆含量的方法,得到Ct值对转基因食品百分含量对数值的标准曲线方程式为Y=-2.819 4X+27.185 5,相关系数值R2为0.999 4。12种市售大豆及制品中五香茶干、豆奶粉、素鸡、热狗肠、薄白叶检测出转基因成分含量分别为2.46%、6.26%、13.95%、0.48%、0.66%,其余7份样品为阴性。结论建立的TaqMan探针实时荧光定量PCR法能够快速地进行转基因大豆含量的检测,并可应用于市售转基因食品的检测。

Objective To establish method of TaqMan PCR for detection of genetically modified soybean, and apply it to the market selling GMO＇s quantitation. Methods RoundUp ReadyTM of genetically modified soybean （Fluka） of 0,0.1% ,0.5% , 2.0% and 5.0% were used as standard substance. By using specific TaqMan probe sequence 5＇ （FAM） - cccactatccttcgcaagaccct - 3＇ （TAMRA） and primer se- quences F： 5＇ - tgatgtgatatctccactgacg - 3＇, R： 5＇ - tgtatcccttgagccatgttgt - 3 ＇ to amplify the exogenous gene CP4EPSPS, we established a standard curve for quantitative detection of genetically modified soybean. Twelve soybean products selling in the market were quantitatively detected. Results The method of TaqMan PCR for detection of genetically modified soybean was successfully established. The equation of standard curve was Y = -2. 819 4X ＋ 27. 185 5 and relative coefficiency R^2 was 0. 999 4. The contents of 5 kinds of genetically modified foods selling in the market were 2.46%, 6.26%, 13.95%, 0.48% 0.66% ,respectivcly. Conclusion This method could quantitative detect genetic＇ally modified soybean rapidly, and could be used to analysis the content of genetically modified foods selling in the market.