Abstract
An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100–1000 cfu ml−1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 106 and 107 cells ml−1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.
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Biagini RE, Schlottmann SA, Sammons DL, Smith JP, Snawder JC, Striley CA, MacKenzie BA, Weissman DN (2003) Method for simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides. Clinical and Diagnostic Laboratory Immunology 10: 744–750
Biagini RE, Sammons DL, Smith JP, MacKenzie BA, Striley CAF, Semenova V, Steward-Clark E, Stamey K, Freeman AE, Quinn CP, Snawder JE (2004) Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins. Clinical and Diagnostic Laboratory Immunology 11: 50–55
Burkholder WH (1953) Genus VI. Erwinia Winslow et al. 1917. In Bergey’s Manual of Determinative Bacteriology, 7th edn., Williams and Wilkins, Baltimore, MD, USA, pp. 349–359
Dasso J, Lee J, Bach H, Mage RG (2002) A comparison of ELISA and flow microsphere-based assays for quantification of immunoglobulins. Journal of Immunological Methods 263: 23–33
De Boer SH, Ward LJ (1995) PCR detection of Erwinia carotovora subsp. atroseptica associated with potato tissue. Phytopathology 85: 854–858
De Boer SH, Copeman RJ, Vruggink H (1979) Serogroups of Erwinia carotovora potato strains determined with diffusible somatic antigens. Phytopathology 69: 316–319
De Boer SH, McNaughton ME (1987) Monoclonal antibodies to the lipopolysaccharide of Erwinia carotovora subsp. atroseptica serogroup I. Phytopathology 77: 828–832
Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacobson JW (2003) Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. Journal of Microbiological Methods 53: 245–252
DuPont NC, Wang K, Wadhwa PD, Culhane JF, Nelson EL (2005) Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology 66: 175–191
Dye DW (1969) A taxonomic study on the genus Erwinia: II. the “carotovora” group. New Zealand Journal of Science 12: 81–97
Earley MC, Vogt RF Jr, Shapiro HM, Mandy FF, Kellar KL, Bellisario R, Pass KA, Marti GE, Stewart CC, Hannon WH (2002) Report from a workshop on multianalyte microsphere assays. Cytometry 50: 239–242
Gardan L, Gouy C, Christen R, Samson R (2003) Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. International Journal of Systematic and Evolutionary Microbiology 53: 381–391
Gorris MT, Alarcon B, Lopez MM, Cambra M (1994) Characterization of monoclonal antibodies specific for Erwinia carotovora subsp. atroseptica and comparison of serological methods for its sensitive detection on potato tubers. Applied and Environmental Microbiology 60: 2076–2085
Hyman LJ, Wallace A, Lopez MM, Cambra M, Gorris MT, Pérombelon MCM (1995) Characterization of monoclonal antibodies against Erwinia carotovora subsp. atroseptica Serogroup I: Specificity and epitope analysis. Journal of Applied Bacteriology 78: 437–444
Janse JD, Ruissen MA (1988) Characterization and classification of Erwinia chrysanthemi strains from several hosts in the Netherlands. Phytopathology 78: 800–808
Kellar KL, Iannone MA (2002) Multiplexed microsphere-based flow cytometric assays. Experimental Hematology 30: 1227–1237
Kellar KL (2003) Applications of multiplexed fluorescent microsphere-based assays to studies of infectious disease. Journal of Clinical Ligand Assay 26: 76–86
McBride MT, Gammon S, Pitesky M, O’Brien TW, Smith T, Aldrich J, Langlois RG, Colston B, Venkateswaran KS (2003) Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents. Analytical Chemistry 75: 1924–1930
Meneley JC, Stanghellini ME (1976) Isolation of soft-rot Erwinia spp. from agricultural soils using an enrichment technique. Phytopathology 66: 367–370
Nassar A, Darrasse A, Lemattre M, Kotoujansky A, Dervin C, Vedel R, Bertheau Y (1996) Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Applied and Environmental Microbiology 62: 2228–2235
Perminow JI (1997) Potato blackleg, caused by Erwinia carotovora subspecies atroseptica, in Norway. Dept. of Plant Pathology. SLU, ISSN 0802–3220, No 12
Pérombelon MCM, Kelman A (1980) Ecology of the soft rot erwinias. Annual Review of Phytopathology 18: 361–387
Rao RS, Visuri SR, McBride MT, Albala JS, Matthews DL, Coleman MA (2004) Comparison of multiplexed techniques for detection of bacterial and viral proteins. Journal of Proteome Research 3: 736–742
Samson R, Poutier F, Sailly M, Jouan B (1987) Caractérisation des Erwinia chrysanthemi isolées de Solanum tuberosum et d’autres plantes-hôtes selon les biovars et sérogroupes. Bulletin OEPP/EPPO Bulletin 17: 11–16
Samson R, Legendre JB, Christen R, Saux MFL, Achouak W, Gardan L (2005) Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953; Brenner et al. 1973) and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. International Journal of Systematic and Evolutionary Microbiology 55: 1415–1427
Selby C (1999) Interference in immunoassay. Annual Clinical Biochemistry 36: 704–721
Śledź W, Jafra S, Waleron M, Łojkowska E (2000) Genetic diversity of Erwinia carotovora strains isolated from infected plants growing in Poland. EPPO Bulletin 30: 413–420
Spiro A, Lowe M (2002) Quantitation of DNA sequences in environmental PCR products by a multiplexed, bead-based method. Applied and Environmental Microbiology 68: 1010–1013
Van Beckhoven JRCM, Van Hoof R, De Raaij-Wieringa G, Bonants P, Van der Wolf J, De Haan E, and Van den Bovenkamp G (2001) Survey naar serogroepen van Erwinia carotovora subsp. atroseptica en E. chrysanthemi in aardappel in Nederland. Internal Report, Plant Research International, 21 pp.
Van der Wolf JM, Gussenhoven GC (1992) Reactions of saprophytic bacteria from potato peel extracts and plant pathogenic bacteria in ELISA with antisera to Erwinia chrysanthemi (serogroup O1Ha). Netherlands Journal of Plant Pathology 98: 33–44
Van der Wolf JM, van Beckhoven JRCM, de Boef E, Roozen NJM (1993) Serological characterization of fluorescent Pseudomonas strains cross-reacting with antibodies against Erwinia chrysanthemi. Netherlands Journal of Plant Pathology 99: 51–60
Van der Wolf JM, Hyman LJ, Jones DAC, Grevesse C, Van Beckhoven JRCM, Van Vuurde JWL, Pérombelon MCM (1996) Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR. Journal of Applied Bacteriology 80: 487–495
Van Vuurde JWL, Roozen NJM (1990) Comparison of immuno colony-staining in media, selective isolation on pectate medium, ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in cattle manure slurry. Netherlands Journal of Plant Pathology 96: 75–89
Vernon-Shirley M, Burns R (1992) The development and use of monoclonal antibodies for detection of Erwinia. Journal of Applied Bacteriology 72: 97–102
Vignali DA (2000) Multiplexed particle-based flow cytometric assays. Journal of Immunological Methods 243: 243–255
Vruggink H, De Boer SH (1978) Detection of Erwinia carotovora var. atroseptica in potato tubers with immunofluorescence following induction of decay. Potato Research 21: 225–229
Acknowledgements
We are indebted to Henk Velvis (HZPC Research, Metslawier, The Netherlands) for providing potato samples and data on ELISA results. We also thank Mrs L.J. Hyman (SCRI, Invergowrie, Scotland, UK) for her editorial work. The work was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality (programme DWK397).
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Peters, J., Sledz, W., Bergervoet, J.H.W. et al. An enrichment microsphere immunoassay for the detection of Pectobacterium atrosepticum and Dickeya dianthicola in potato tuber extracts. Eur J Plant Pathol 117, 97–107 (2007). https://doi.org/10.1007/s10658-006-9068-6
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DOI: https://doi.org/10.1007/s10658-006-9068-6