Evaluation of a PCR kit for the detection ofErwinia carotovora subsp.atroseptica on potato tubers |
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Authors: | Dominique Frechon Pascale Exbrayat Valerie Helias Lizbeth J. Hyman B. Jouan P. Llop Maria M. Lopez Nicole Payet M. C. M. Pérombelon I. K. Toth José R. C. M. van Beckhoven J. M. van der Wolf Y. Bertheau |
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Affiliation: | (1) Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, 16 rue Claude Bernard, 75231 Paris, France;(2) Institut National de la Recherche Agronomique, BP 9, 35653 Le Rheu, France;(3) Scottish Crop Research Institute, Invergowrie, DD2 5DA Dundee, UK;(4) Instituto Valenciano Investigaciones Agrarias, 46113 Moncada, Valencia, Spain;(5) Instituut voor Planteziektenkundig Onderzoek DLO, P.O. Box 9060, 6700 GW Wageningen, The Netherlands |
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Abstract: | Summary A PCR-based kit, ProbeliaTM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3×102 to 1.5×103 cells ml−1). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0×101 to 6.2×103 cells ml−1 peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method. |
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Keywords: | blackleg ELISA DNA |
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