Comparison of DNA extraction methods to detect Pseudogymnoascus destructans in bat skin swabs, guano, and environmental swabs.

Abstract

National surveillance for Pseudogymnoascus destructans-the fungus that causes white-nose syndrome in bats-relies on methods optimized for highly sensitive detection of the pathogen. However, comparisons of methods that inform assay functionality are sparse. We used a combination of conidia-spiking experiments and analysis of field-collected samples to compare methods for DNA extraction from bat guano, skin swabs, and environmental swabs for subsequent detection of P. destructans DNA via quantitative real-time PCR (qPCR). We evaluated 2 methods for all sample types and a third method for guano samples. We created conidia-inoculated guano samples from 3 guano sources, as well as conidia-inoculated environmental swabs using sediment from 7 bat hibernacula. Detection of P. destructans from environmental swabs was 2.94 times more likely from a purification-free method of DNA extraction, with up to 9.75 times more target DNA detected on average than a magnetic bead purification method, as evidenced by both laboratory-spiked and field-collected samples. We observed a similar result for skin swabs collected from P. destructans-infected bat carcasses, with a 31.7-fold increase in the amount of target DNA detected on average. Between the same 2 extraction methods, a 3.3-fold difference was found in the odds of detecting P. destructans from guano, but a third method designed for DNA extraction from soil had 12.6-41.8 times higher odds of detection. Our study further optimizes DNA extraction methods for the detection of P. destructans from environmental swabs and offers a detailed comparison of extraction methods used for detection from bat skin swabs and guano.