Development and Application of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Diagnosis of the Bat White-Nose Disease Fungus Pseudogymnoascus destructans.

Pseudogymnoascus destructans (= Geomyces destructans) is a psychrophilic filamentous fungus that causes White-Nose Disease (WND; the disease associated with White-Nose Syndrome, WNS) in hibernating bats. The disease has caused considerable reductions in bat populations in the USA and Canada since 2006. Identification and detection of the pathogen in pure cultures and environmental samples is routinely based on qPCR or PCR after DNA isolation and purification. Rapid and specific direct detection of the fungus in the field would strongly improve prompt surveillance, and support control measures. Based on the genes coding for ATP citrate lyase1 (acl1) and the 28S-18S ribosomal RNA intergenic spacer (IGS) in P. destructans, two independent LAMP assays were developed for the rapid and sensitive diagnosis of the fungus. Both assays could discriminate P. destructans from 159 tested species of filamentous fungi and yeasts. Sensitivity of the assays was 2.1 picogram per reaction (pg/rxn) and 21 femtogram per reaction (fg/rxn) for the acl1 and IGS based assays, respectively. Moreover, both assays also work with spores and mycelia of P. destructans that are directly added to the master mix without prior DNA extraction. For field-diagnostics, we developed and tested a field-applicable version of the IGS-based LAMP assay. Lastly, we also developed a protocol for preparation of fungal spores and mycelia from swabs and tape liftings of contaminated surfaces or infected bats. This protocol in combination with the highly sensitive IGS-based LAMP-assay enabled sensitive detection of P. destructans from various sources.