Vol. 61 No. 3 (2022): including the "60th MPU Anniversary Special Section"
Articles

Nanoplate digital PCR assays for detection and quantification of Xylella fastidiosa

Alessandro PASSERA
Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, University of Milan, Milan
Valentina GROSSO
Department of Biotechnology, University of Verona, Verona
Niccolò MIOTTI
Department of Agricultural and Environmental Sciences – ProDepartment of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, University of Milan, Milan
Marzia ROSSATO
Department of Biotechnology, University of Verona, Verona
Francesca GAFFURI
Laboratorio del Servizio Fitosanitario, Servizio Fitosanitario Regione Lombardia, Fondazione Minoprio, Vertemate con Minoprio
Paola CASATI
Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, University of Milan, Milan
Massimo DELLEDONNE
Department of Biotechnology, University of Verona, Verona
Piero Attilio Bianco
Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, University of Milan, Milan
Published January 13, 2023
Keywords
  • qPCR,
  • Nerium oleander,
  • TaqMan,
  • EvaGreen,
  • dPCR
How to Cite
[1]
A. PASSERA, “Nanoplate digital PCR assays for detection and quantification of Xylella fastidiosa”, Phytopathol. Mediterr., vol. 61, no. 3, pp. 489-503, Jan. 2023.

Funding data

Abstract

Xylella fastidiosa is a fastidious Gram-negative bacterium that is associated with several important plant diseases, and is regulated as a quarantine pest in many countries where strategies are implemented to prevent its introduction and spread. To enact efficient quarantine measures, effective and early detection of the pathogen are essential, especially because global trade of goods increases the risks of introduction of alien pathogens. this study aimed to adapt two qPCR-based diagnostic methods (SYBR Green and Probe based qPCR), already in use to detect X. fastidiosa, for use with a nanoplate based digital PCR assay. Detection of the pathogen using the two digital PCR assays (EvaGreen- and Probe-based) was similar to standard qPCR, giving 100% sensitivity, specificity, and accuracy, while providing accurate absolute quantification of the pathogen when using experimental samples that had low concentrations of host DNA. Using undiluted plant DNA added with low concentrations of X. fastidiosa, only the TaqMan method maintained satisfactory performance and quantification, and is therefore preferred. These results are a first step demonstrating the usefulness of nanoplate-based digital PCR for detection of plant pathogens, which allows greater throughput than qPCR, reducing the time and cost of diagnostic assays.

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