Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers |
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| Authors: | Palmira De Bellis Leonardo Schena Corrado Cariddi |
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| Institution: | (1) Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy;(2) Department of Plant Protection and Applied Microbiology, University of Bari, Via Amendola 165/a, 70126 Bari, Italy;(3) Present address: Department of Management of Agricultural and Forest Systems, Mediterranean University, Località Feo di Vito, 89122 Reggio Calabria, Italy |
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| Abstract: | 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. |
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| Keywords: | Fire Blight Molecular detection Nested PCR Plasmid pEA29 Scorpion-PCR (duplex format) |
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