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.
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Abbreviations
- CRMs:
-
Certified reference materials
- CV:
-
Coefficients of variation
- GMO:
-
Genetically modified organism
- LODs:
-
Limits of detection
- LOQs:
-
Limits of quantitation
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Acknowledgment
This study is supported by the National Major Project of Breeding for Genetically Modified Organism in China (no. 2009ZX 08012-012B).
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Qingfeng Guan and Xiumin Wang contributed equally to this paper.
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Guan, Q., Wang, X., Teng, D. et al. Construction of a Standard Reference Plasmid for Detecting GM Cottonseed Meal. Appl Biochem Biotechnol 165, 24–34 (2011). https://doi.org/10.1007/s12010-011-9230-2
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DOI: https://doi.org/10.1007/s12010-011-9230-2