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Evaluation and validation of a loop-mediated isothermal amplification test kit for detection of Hymenoscyphus fraxineus

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Abstract

Hymenoscyphus fraxineus (anamorph Chalara fraxinea) is the ascomycete fungus which causes ash dieback, a potentially lethal disease of Fraxinus excelsior in Europe. Isolation and culturing of H. fraxineus is time consuming and there is a need for rapid, specific diagnostic tools to assist in the deployment of appropriate phytosanitary measures. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method which can be used for on-site diagnosis. OptiGene have recently released a kit for rapid in planta detection of H. fraxineus using LAMP. The performance of the kit was evaluated for use in the laboratory and in the field in a comparison with real-time PCR, and the kit was also validated according to EPPO standard 7/98. The analytical sensitivity of the kit was found to be 7 pg of DNA extracted from pure culture. The kit was rapid, with an average time to positive of 15.5 min for field samples. A comparison of real-time PCR and LAMP was carried out in the laboratory and in the field. The diagnostic sensitivity and specificity of the LAMP kit in the laboratory were 90.2% and 98.0% respectively, and 98.3% and 88.6% in the field. Positive and negative predictive values and likelihood ratios were also calculated and considered in relation to the prevalence of the disease. The kit was found to be a useful diagnostic tool which can be applied in the field.

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Acknowledgements

The authors gratefully acknowledge the assistance of Forestry Commission England Plant Health Officers.

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Authors and Affiliations

  1. Fera Science Ltd, NAFIC, Sand Hutton, York, YO41 1LZ, UK

    Catherine Harrison, Jenny Tomlinson, Sioban Ostoja-Starzewska & Neil Boonham

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  1. Catherine Harrison
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  2. Jenny Tomlinson
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  3. Sioban Ostoja-Starzewska
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  4. Neil Boonham
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Correspondence to Catherine Harrison.

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Harrison, C., Tomlinson, J., Ostoja-Starzewska, S. et al. Evaluation and validation of a loop-mediated isothermal amplification test kit for detection of Hymenoscyphus fraxineus . Eur J Plant Pathol 149, 253–259 (2017). https://doi.org/10.1007/s10658-017-1179-8

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  • DOI: https://doi.org/10.1007/s10658-017-1179-8

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