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

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 tec...

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Bibliographic Details
Published in:Folia microbiologica 2010-05, Vol.55 (3), p.239-244
Main Authors: Kokošková, B., Mráz, I., Fousek, J.
Format: Article
Language:English
Subjects:
Actinomycetales - classification
Actinomycetales - genetics
Actinomycetales - immunology
Actinomycetales - isolation & purification
Applied Microbiology
Bacteria
Bacteriological Techniques - methods
Biomedical and Life Sciences
Clavibacter michiganensis
Comparative studies
Cross Reactions
Curtobacterium flaccumfaciens
DNA Primers - genetics
Environmental Engineering/Biotechnology
Enzyme-Linked Immunosorbent Assay - methods
Immunology
Life Sciences
Lycopersicon esculentum
Lycopersicon esculentum - microbiology
Microbiology
Molecular biology
Nucleic Acid Hybridization - methods
Pectobacterium
Plant Diseases - microbiology
Polymerase Chain Reaction - methods
Rahnella aquatilis
Sensitivity analysis
Sensitivity and Specificity
Temperature
Time Factors
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Summary: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 10 5 CFU/mL and 10 3 CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10 3 CFU/mL with our designed primers and 104 CFU/mL with Dreier’s primer pair.
ISSN:0015-5632
1874-9356
DOI:10.1007/s12223-010-0035-4