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Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events

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Abstract

Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90 %, and the squared regression coefficients (R2) were more than 0.999. The quantification bias varied from 0.21 % to 19.29 %, and the relative standard deviations were from 1.08 % to 9.84 % in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

One multi-targets plasmid, pSOY, that can be used as calibrator in the detection of the five GM soybean Events

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Acknowledgments

This work was supported by the National Transgenic Plant Special Fund (2014ZX08012-003 and 2014ZX08012001-007), the National Natural Science Foundation of China (31471670), and Program for New Century Excellent Talents in University.

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    Authors and Affiliations

    1. Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

      Liqun Pi, Yiwei Cao, Canhua Wang & Litao Yang

    2. Technical Center for Animal, Plant, and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau of China, No. 1208, Minsheng Road, Shanghai, 200135, China

      Xiang Li & Liangwen Pan

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    1. Liqun Pi
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    2. Xiang Li
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    3. Yiwei Cao
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    4. Canhua Wang
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    Correspondence to Litao Yang.

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    Liqun Pi and Xiang Li contributed equally to this work.

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    Pi, L., Li, X., Cao, Y. et al. Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events. Anal Bioanal Chem 407, 2877–2886 (2015). https://doi.org/10.1007/s00216-015-8517-5

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    • DOI: https://doi.org/10.1007/s00216-015-8517-5

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