Validation of a modified CTAB method for DNA extraction from protein-rich maize feedstuffs

Abstract

The detection of genetically modified organisms in feedstuffs using PCR methods rely on the yield and quality of the extracted genomic plant DNA. The used extraction methods must be able to provide DNA of sufficient amounts of non-fragmented DNA that is free of PCR inhibitors. This article describes the comparison of methods for the extraction of DNA from maize gluten as an example for a protein-rich corn-based feedstuff sample and the following validation of two selected methods. These selected methods are based on lysis by a cetyltrimethylammonium bromide buffer followed by precipitation of genomic DNA and on a kit-based procedure. To calculate the amount of amplifiable DNA copies and the PCR efficiency in the extracts, the crossing points of the real-time PCR curves were used. The comparison of different extraction methods showed that the two selected methods yielded the highest amounts of amplifiable DNA based on the amplification of the maize specific reference gene hmg by real-time TaqMan PCR. Additionally, approaches to increase DNA yield or concentration by variation of the protocol are discussed. Both validation studies were conducted by the working group “PCR analytics in feed” of the Association of German Agricultural and Research Institutes.

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Acknowledgements

We want to thank all laboratories that participated in the ring trials: Hessian State Laboratory (Kassel, Germany), Thuringian State Institute for Agriculture (Jena, Germany), Wessling GmbH (Altenberge, Germany), Austrian Agency for Health and Food Safety, Institute for Food Safety (Wien, Austria), National Reference Laboratory for Animal Protein in Feed, German Federal Institute for Risk Assessment (Berlin, Germany), National Reference Laboratory for Genetically Modified Organisms, Federal Office of Consumer Protection and Food Safety (Berlin, Germany), Bavarian Health and Food Safety Authority (Oberschleissheim, Germany), Chemical and Veterinary Investigatory Office Rhein-Ruhr-Wupper (Krefeld, Germany), Chemical and Veterinary Investigatory Office OWL (Detmold, Germany), Institute for Hygiene and Environment (Hamburg, Germany), Saarland State Office for Consumer Protection (Saarbrücken, Germany). LUFA Speyer (Speyer, Germany), Biolytix AG (Witterswil, Switzerland). In addition we want to thank Benjamin Pickel (LUFA Speyer, Speyer, Germany), Ole Sindt (State Laboratory Schleswig Holstein, Neumuenster, Germany), and Ulf-Martin Kohlstock (State Agency for Agriculture and Horticulture Sachsen-Anhalt) for critically reading the manuscript. The Federal Office of Consumer Protection and Food Safety (Berlin, Germany) provided the chemicals required for the realization of the ring trials.

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Correspondence to Nele Matthes.

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Matthes, N., Westphal, K., Haldemann, C. et al. Validation of a modified CTAB method for DNA extraction from protein-rich maize feedstuffs. J Consum Prot Food Saf (2020). https://doi.org/10.1007/s00003-020-01285-y

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Keywords

  • DNA extraction
  • Maize gluten
  • Genetically modified organism
  • Real-time PCR
  • Limit of quantification