title: Construction of a standard reference plasmid for detecting GM cottonseed meal.
abstract: 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 intra-run 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.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, People's Republic of China.
authors.forename: Qingfeng
authors.initials: Q
authors.lastname: Guan
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Yalin
authors.initials: Y
authors.lastname: Yang
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.forename: Qingqiang
authors.initials: Q
authors.lastname: Yin
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D003369:Cottonseed Oil / Q000737:chemistry*; D003368:Gossypium / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards
chemical_list: D003369:Cottonseed Oil; D010940:Plant Proteins