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Assessment of a new qPCR tool for the detection and identification of the root-knot nematode Meloidogyne enterolobii by an international test performance study

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Abstract

Rapid and reliable tools for detection and identification of plant parasitic nematodes are needed to prevent the introduction and spread of quarantine nematodes. A fast and simple DNA extraction method for target nematodes in nematode suspensions obtained from soil samples and a new quantitative real-time PCR method (qPCR) for the specific detection, identification and potential quantification of M. enterolobii were tested in an inter-laboratory comparison (ring test) to allow for a thorough evaluation of these molecular diagnostic tools. A test performance study involving seven laboratories was conducted to validate the developed protocols and to identify possible difficulties when implemented by diagnostic laboratories or national reference centers. Validation included test performance in terms of accuracy, analytical specificity, analytical sensitivity, repeatability, and reproducibility as defined by European Plant Protection Organization (EPPO) standard PM7/98. All positive and negative results for detection, identification and specificity were consistent between different laboratories despite different equipment used. Accuracy of real-time PCR was 100 % because test results and accepted reference values were in agreement. Analytical sensitivity results also matched between laboratories independent of the equipment used. The smallest amount of target DNA tested, two second-stage juveniles of M. enterolobii in a background of 500 non-target nematodes, was reliably detected by all labs. In addition, the repeatability and reproducibility of test results between laboratories was 100 %, even at the limit of detection. Thus, the inter-laboratory comparison showed the robustness of the developed methods and confirmed the in-house validation data.

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Acknowledgments

This research project was performed within the EUPHRESCO II framework, project “Development and validation of innovative diagnostic tools for detection and identification of the quarantine nematode Meloidogyne enterolobii in support of integrated plant protection strategies in the EU member states”, via a non-competitive funding mechanism. We thank all colleagues for sharing nematode populations and the Swiss Federal Office of Agriculture as well as the Belgian Federal Agency for the Safety of the Food Chain for financial support.

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Authors and Affiliations

  1. Agroscope, Institute for Plant Production Sciences, IPS, Schloss 1, 8820, Waedenswil, CH, Switzerland

    Andrea Braun-Kiewnick & Sebastian Kiewnick

  2. ILVO, Plant-Crop Protection, Nematology, Burg. Van Gansberghelaan 96, 9820, Merelbeke, Belgium

    Nicole Viaene

  3. ANSES, Plant Health Laboratory, Domaine de la Motte au Vicomte BP 35327, 35653, Le Rheu Cedex, France

    Laurent Folcher, Fabrice Ollivier & Géraldine Anthoine

  4. JKI – Julius Kühn-Institut/Plant Health Institute, Messeweg 11/12, D-38104, Braunschweig, Germany

    Björn Niere

  5. FERA - The Food & Environment Research Agency, Sand Hutton, UK-York, YO41 1LZ, UK

    Melanie Sapp

  6. Div. Nematology, National Plant Protection Organization, National Reference Center (NRC), Geertjesweg 15, 6700, HC, Wageningen, Netherlands

    Bart van de Vossenberg

  7. Faculty Agricultural Sciences and Technology, University of Nigde, Nigde, 51240, Turkey

    Halil Toktay

Authors
  1. Andrea Braun-Kiewnick
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  2. Nicole Viaene
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  3. Laurent Folcher
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  4. Fabrice Ollivier
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  5. Géraldine Anthoine
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  7. Melanie Sapp
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  8. Bart van de Vossenberg
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  9. Halil Toktay
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  10. Sebastian Kiewnick
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Correspondence to Sebastian Kiewnick.

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Braun-Kiewnick, A., Viaene, N., Folcher, L. et al. Assessment of a new qPCR tool for the detection and identification of the root-knot nematode Meloidogyne enterolobii by an international test performance study. Eur J Plant Pathol 144, 97–108 (2016). https://doi.org/10.1007/s10658-015-0754-0

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  • DOI: https://doi.org/10.1007/s10658-015-0754-0

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