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Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis

Folia Microbiologica volume 55, pages 239–244 (2010)Cite this article

Abstract

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 °C for Dreier’s and 10 s at 69 °C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 105 CFU/mL and 103 CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 103 CFU/mL with our designed primers and 104 CFU/mL with Dreier’s primer pair.

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Abbreviations

Cmm :

Clavibacter michiganensis subsp. michiganensis

ELISA:

enzyme-linked immunosorbent assay

IFAS:

indirect fluorescent antibody stain

MAb:

monoclonal antibody

PAb:

polyclonal antibody

PBS:

phosphate-buffered saline

PCR:

polymerase chain reactions

PFGE:

pulse-field gel electrophoresis

PTA-ELISA:

plate-trapped enzyme-linked immunosorbent assay

rep-PCR:

repetitive sequence-based polymerase chain reactions

SA:

slide agglutination

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Affiliations

  1. Laboratory of Diagnostics and Epidemiology of Microorganisms, Plant Medicine Division, Crop Research Institute, 161 06, Prague 6, Czech Republic

    B. Kokošková

  2. Department of Plant Virology, Institute of Plant Molecular Biology, Biological Centre of the Academy of Sciences of the Czech Republic, 370 05, České Budějovice, Czech Republic

    I. Mráz & J. Fousek

Authors
  1. B. Kokošková
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  2. I. Mráz
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  3. J. Fousek
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Correspondence to B. Kokošková.

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Kokošková, B., Mráz, I. & Fousek, J. Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis . Folia Microbiol 55, 239–244 (2010). https://doi.org/10.1007/s12223-010-0035-4

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  • DOI: https://doi.org/10.1007/s12223-010-0035-4

Keywords

  • Tomato Seed
  • Ralstonia Solanacearum
  • Plant Pathogenic Bacterium
  • Bacterial Cell Suspension
  • Bacterial Canker