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Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects

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Abstract

Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid : (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.

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Acknowledgements

This research was done within a Belgian research project (S-6140) financed by DG4 and DG6 of the former Belgian Federal Ministry of Agriculture. We are grateful to Aurélie Hosselet (HECE Fleurus) for her technical help and Nicole Wellens (Isogen Life Sciences, De Meern, the Netherlands) for advice with pyrosequencing.

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Authors and Affiliations

  1. Department of Quality of Agricultural Products, Walloon Agricultural Research Center (CRA-W), 24, chaussée de Namur, 5030, Gembloux, Belgium

    Frédéric Debode, Aline Marien, Eric Janssen & Gilbert Berben

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  1. Frédéric Debode
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  2. Aline Marien
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  3. Eric Janssen
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  4. Gilbert Berben
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Correspondence to Gilbert Berben.

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Debode, F., Marien, A., Janssen, E. et al. Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects. Anal Bioanal Chem 396, 2151–2164 (2010). https://doi.org/10.1007/s00216-009-3396-2

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  • DOI: https://doi.org/10.1007/s00216-009-3396-2

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