Development of a Loop-Mediated Isothermal Amplification Assay as an Early-Warning Tool for Detecting Emerald Ash Borer (Coleoptera: Buprestidae) Incursions.

The emerald ash borer (EAB), Agrilus planipennis (Fairmaire), is the most destructive invasive insect species of ash (Fraxinus spp.) in North America. An accurate method for early detection of this noxious insect pest is indispensable to providing adequate warning of A. planipennis infestation. A loop-mediated isothermal amplification (LAMP) assay (EAB-LAMP) was developed based on mitochondrial cytochrome c oxidase subunit I (COI) gene. The EAB-LAMP required only 30 min at 65°C to amplify A. planipennis DNA from specimens collected from geographically distinct locations. There was no cross-reactivity with other Agrilus and insect species. The developed EAB-LAMP differentially detected traces of A. planipennis genome (COI) within frass from various Fraxinus species. EAB-LAMP was also able to distinguish among A. planipennis DNA and other Agrilus species and nontarget insect species in trap captures. By detecting A. planipennis DNA in two additional trap captures (in situ), the EAB-LAMP was more sensitive and reliable than visual inspection. We tested the quantitative nature of the assay by evaluating pooled trap samples and demonstrated that the EAB-LAMP was capable of functioning optimally using a pool size of at least five individual trap samples. This potentially circumvents the need to perform large-scale individual analysis for processing trap samples. Considering its performance, specificity, sensitivity, and repeatability, the developed EAB-LAMP could be a valuable tool to support strategy and operation of large-scale surveillance for A. planipennis and could profitably be used in routine monitoring programs for effective management of A. planipennis.