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Development of dual-labeled PNA probe-based fluorescence melting curve analysis as molecular diagnostic tool for Erwinia amylovora and E. pyrifoliae

Moon Nam: Xenotype Company Limited

<div>Due to the recent report of fire blight outbreaks of apples and pears in Anseong, South Korea in 2015, the development of effective diagnostic methods for bacterial pathogens of fire blight is becoming more important. The diagnosis of <em>E. amylovora</em> has been developed in various ways, such as normal PCR assay, Taqman assay, SYBR Green assay, and lateral-flow immunographic strip methods. In this study, we identified genome-wide single nucleotide polymorphism (SNP) markers using smart sequencing by PACBIO to discriminate between <em>E. amylovora</em> and <em>E. pyrifoliae</em> and used the markers in epidemiological survey. Nine candidate SNP markers were selected and the three of them were evaluated for the performance of dual-labeled peptide nucleic acid (PNA) probe-based fluorescence melting curve analysis (FMCA) as a detection tool of <em>E. amylovora </em>and <em>E. pyrifoliae</em>. A total of <em>E. amylovora</em> isolates from various regions and related <em>Erwinia </em>sp. isolates (<em>E. pyrifoliae</em> and <em>E. rhapontici</em>) were investigated. Compared with the conventional methods, PNA probe-based FMCA showed improved sensitivity and more reliable diagnostic performance without false-positive results. The newly developed diagnostics method, PNA probe-based FMCA will provide more reliability and rapidity in confirmation of the <em>E. amylovora</em> in apple and pear trees.</div>