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Construction of a Standard Reference Plasmid for Detecting GM Cottonseed Meal

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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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Authors and Affiliations

  1. Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, People’s Republic of China

    Qingfeng Guan, Xiumin Wang, Da Teng, Yalin Yang, Fang Tian & Jianhua Wang

  2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, People’s Republic of China

    Qingfeng Guan & Qingqiang Yin

  3. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St. Haidian District, Beijing, 100081, People’s Republic of China

    Xiumin Wang, Da Teng, Yalin Yang & Jianhua Wang

Authors
  1. Qingfeng Guan
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  2. Xiumin Wang
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  3. Da Teng
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  4. Yalin Yang
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  5. Fang Tian
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  7. Jianhua Wang
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Corresponding author

Correspondence to Jianhua Wang.

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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

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