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Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

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Abstract

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.

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Acknowledgments

The authors would like to thank Direction Générale de la Consommation, de la Concurrence et de la Répression des Fraudes (DGCCRF) and Direction Générale de l'Alimentation (DGAl) for kindly providing us with samples. This work was financially supported by the European Commission through the Sixth Framework Program, integrated project Co-Extra http://www.coextra.eu; contract FOOD-2005-CT-007158). This is gratefully acknowledged. The authors thank Dr Y. Roussel for critical reading of the manuscript.

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Authors and Affiliations

  1. UMR 1290, INRA, Route de Saint-Cyr, 78000, Versailles, France

    Julien Pansiot, Maher Chaouachi, Laetitia Cavellini, Marcel Romaniuk, Mira Ayadi, Yves Bertheau & Valérie Laval

  2. SPE, INRA, Route de Saint-Cyr, 78000, Versailles, France

    Yves Bertheau

  3. Unité de Biochimie et Interaction Moléculaire 02/UR/09-01, ISBM, Monastir, Tunisia

    Maher Chaouachi

  4. INRA centre de Versailles, Route de Saint-Cyr, 78026, Versailles Cedex, France

    Yves Bertheau

  5. UMR1290 Unité BIOGER-CPP (bat. 13), INRA-AgroParisTech, Avenue Lucien Brétignières, 78850, Thiverval-Grignon, France

    Valérie Laval

Authors
  1. Julien Pansiot
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  2. Maher Chaouachi
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  3. Laetitia Cavellini
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  4. Marcel Romaniuk
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  5. Mira Ayadi
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  6. Yves Bertheau
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  7. Valérie Laval
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Corresponding authors

Correspondence to Yves Bertheau or Valérie Laval.

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Pansiot, J., Chaouachi, M., Cavellini, L. et al. Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes. Eur Food Res Technol 232, 327–334 (2011). https://doi.org/10.1007/s00217-010-1394-4

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  • DOI: https://doi.org/10.1007/s00217-010-1394-4

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