Real-time Scorpion-PCR detection and quantification of Erwinia amylovora on pear leaves and flowers
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A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 104 CFU ml−1 in Scorpion-PCR and 2.8 × 102 CFU ml−1 in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.
KeywordsFire Blight Molecular detection Nested PCR Plasmid pEA29 Scorpion-PCR (duplex format)
This work was supported by a grant from the University of Bari: ‘Epidemiology and genetics of plant-pathogen microrganisms’. We thank J.D. Janse Jr. at Department of Bacteriology, Plant Protection Service, Wageningen, The Netherlands, C. Bazzi at Department of Agroenvironmental Sciences and Technologies, University of Bologna, Italy, M. Scortichini at Fruit Tree Research Institute, Rome, Italy, and L. Corazza at Plant Pathology Research Institute, Rome, Italy, for kindly supplying bacterial isolates.
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