Methods for sample preservation and DNA extraction affect recovery of Pseudogymnoascus destructans from conidia-spiked swabs

Abstract

Samples collected to conduct pathogen surveillance for the causative fungus of bat white-nose syndrome, Pseudogymnoascus destructans (Pd), are analyzed by multiple laboratories in the United States and Canada using various sample storage and molecular techniques. Comparative analyses among methods have been lacking, and discordant results from field samples can be confusing for wildlife managers. Using Pd-inoculated sterile swabs (10³–10⁵ conidia), we evaluated Pd DNA recovery via qPCR following different storage conditions and subsequent DNA extraction in 2 commonly used methods. Method B resulted in significantly greater quantities of recovered DNA regardless of storage duration (3 d, 1 mo, 3 mo), storage temperature (ambient temperature, 4ºC, −20ºC), and whether samples were stored with or without RNAlater preservative. Limit of detection (LOD) analyses indicated that method B provided Pd detection from 75% of extracted samples inoculated with just 4 conidia compared with 20% for method A; theoretical LODs, based on a 95% detection rate, were 10.0 conidia (95% CI: 5.7, 14.3) and 537 conidia (95% CI: 176, 897) for a single qPCR replicate, respectively. Our results provide an initial step in validating methods to support surveillance efforts for early detection of fungal invasion, enabling laboratories to select an approach that will preserve sample integrity, benefit Pd detectability, and reduce variability in DNA recovery across a wide range of conidia concentrations.