Construction of a standard reference plasmid for detecting GM cottonseed meal

文献类型： 外文期刊

第一作者： Wang, Xiumin

作者： Wang, Xiumin;Teng, Da;Yang, Yalin;Wang, Jianhua;Guan, Qingfeng;Wang, Xiumin;Teng, Da;Yang, Yalin;Tian, Fang;Wang, Jianhua;Guan, Qingfeng;Yin, Qingqiang

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关键词： Bt cottonseed meal;Construction;Real-time PCR;Standard plasmid

期刊名称：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY （ 影响因子：2.926； 五年影响因子：2.685 ）

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收录情况： SCI

摘要： A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD?19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intrarun coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.

分类号： Q5

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