Construction of a standard reference plasmid for detecting GM cottonseed meal

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.