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Merighi M, Sandrini A, Landini S, et al. Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29. Plant Dis. 2000;84(1):49-54doi: 10.1094/PDIS.2000.84.1.49.
Merighi, M., Sandrini, A., Landini, S., Ghini, S., Girotti, S., Malaguti, S., & Bazzi, C. (2000). Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29. Plant disease, 84(1), 49-54. https://doi.org/10.1094/PDIS.2000.84.1.49
Merighi, M, et al. "Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29." Plant disease vol. 84,1 (2000): 49-54. doi: https://doi.org/10.1094/PDIS.2000.84.1.49
Merighi M, Sandrini A, Landini S, Ghini S, Girotti S, Malaguti S, Bazzi C. Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29. Plant Dis. 2000 Jan;84(1):49-54. doi: 10.1094/PDIS.2000.84.1.49. PMID: 30841222.
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Plant Dis. 2000 Jan;84(1):49-54. doi: 10.1094/PDIS.2000.84.1.49.

Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29.

Plant disease

M Merighi, A Sandrini, S Landini, S Ghini, S Girotti, S Malaguti, C Bazzi

Affiliations

  1. Department of Plant Pathology, The Ohio State University, Columbus 43210.
  2. UCI/SCRM Institute of Chemical Sciences, University of Bologna, Italy.
  3. UCI/STAA Institute of Plant Pathology, University of Bologna, Italy.

PMID: 30841222 DOI: 10.1094/PDIS.2000.84.1.49

Abstract

A molecular diagnostic technique (polymerase chain reaction enzyme-linked immunosorbent assay [PCR-ELISA]) for detection of Erwinia amylovora was developed. The protocol is based on the immunoenzymatic determination of PCR products. For in vitro amplification, we used previously published primers able to detect the cryptic plasmid pEA29, which is ubiquitous in E. amylovora. Amplicons were labeled with 11-digoxigenin (DIG)-dUTP during the amplification reaction, captured by hybridization to a biotinylated oligonucleotide in streptavidin-coated ELISA microplates, and then detected with anti-DIG-Fab'-peroxidase conjugated antibodies. The specificity of the assay was verified using E. amylovora strains from different host plants and geographical origins in addition to other plant-associated bacteria (either phytopathogenic or saprophytic) belonging to the genera Erwinia, Pseudomonas, and Agrobacterium. In detection threshold experiments with pure cultures, as few as 30 and 3 CFU/reaction tube were detected when the ABTS (colorimetric) and ECL (chemiluminescent) detection assays, respectively, were used. PCR-ELISA coupled with chemiluminescent detection was able to detect as few as 4 × 10

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