title: Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.
abstract: Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.
authors.affiliation: Agroscope Changins-Wädenswil Research Station ACW, Plant Protection Division, CH-8820 Wädenswil, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bühlmann
authors.forename: Joël F
authors.initials: JF
authors.lastname: Pothier
authors.forename: Fabio
authors.initials: F
authors.lastname: Rezzonico
authors.forename: Theo H M
authors.initials: TH
authors.lastname: Smits
authors.forename: Michael
authors.initials: M
authors.lastname: Andreou
authors.forename: Neil
authors.initials: N
authors.lastname: Boonham
authors.forename: Brion
authors.initials: B
authors.lastname: Duffy
authors.forename: Jürg E
authors.initials: JE
authors.lastname: Frey
mesh_terms: D000383:Agriculture / Q000379:methods*; D001431:Bacteriological Techniques / Q000379:methods*; D044084:Erwinia amylovora / Q000235:genetics / Q000302:isolation & purification*; D027845:Malus / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D031989:Pyrus / Q000382:microbiology; D012680:Sensitivity and Specificity; D013997:Time Factors