title: Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples.
abstract: The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay.
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI (Verant)U.S. Geological Survey-National Wildlife Health Center, Madison, WI (Bohuski, Lorch, Blehert).
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Verant
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI (Verant)U.S. Geological Survey-National Wildlife Health Center, Madison, WI (Bohuski, Lorch, Blehert).
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Bohuski
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI (Verant)U.S. Geological Survey-National Wildlife Health Center, Madison, WI (Bohuski, Lorch, Blehert).
authors.forename: Jeffery M
authors.initials: JM
authors.lastname: Lorch
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI (Verant)U.S. Geological Survey-National Wildlife Health Center, Madison, WI (Bohuski, Lorch, Blehert) dblehert@usgs.gov.
authors.forename: David S
authors.initials: DS
authors.lastname: Blehert
mesh_terms: D000818:Animals; D001203:Ascomycota / Q000235:genetics / Q000302:isolation & purification*; D002685:Chiroptera / Q000382:microbiology*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004783:Environmental Microbiology; D005656:Fungal Proteins / Q000235:genetics; D009181:Mycoses / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D009656:North America; D009666:Nose / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins
keywords: Bats; DNA extraction; Pseudogymnoascus destructans; quantification; white-nose syndrome