Related Experiment Video
Updated: Jun 2, 2026
Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA
Published on: May 2, 2018
Methods for sample preservation and DNA extraction affect recovery of Pseudogymnoascus destructans from
Kyle G George1, Anne E Ballmann1
1U.S. Geological Survey, National Wildlife Health Center, Madison, WI, USA.
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.
Insights
Method B significantly improves DNA recovery for Pseudogymnoascus destructans (Pd) surveillance. This method enhances Pd detection sensitivity, crucial for early identification of white-nose syndrome in bats.
Area of Science:
- Mycology
- Wildlife Disease Ecology
- Molecular Diagnostics
Background:
- Bat white-nose syndrome, caused by Pseudogymnoascus destructans (Pd), requires effective surveillance.
- Current surveillance relies on diverse lab methods, leading to inconsistent results.
- Comparative analysis of storage and extraction methods for Pd is lacking.
Purpose of the Study:
- To evaluate Pd DNA recovery using two common molecular methods under various storage conditions.
- To determine the optimal method for preserving sample integrity and maximizing Pd detectability.
- To reduce variability in DNA recovery for more reliable surveillance data.
Main Methods:
- Pd-inoculated sterile swabs were subjected to different storage durations (3 days to 3 months) and temperatures (ambient, 4°C, -20°C).
- Samples were stored with or without RNAlater preservative.
- DNA was extracted using two common methods (Method A and Method B) and analyzed by qPCR.
Main Results:
- Method B yielded significantly higher Pd DNA recovery compared to Method A across all storage conditions.
- Method B demonstrated a lower limit of detection, successfully identifying Pd from samples with as few as 4 conidia.
- Theoretical LOD for Method B was 10 conidia, versus 537 conidia for Method A.
Conclusions:
- Method B is superior for Pd DNA recovery, enhancing sensitivity and reliability in surveillance.
- Standardizing methods like Method B will improve the accuracy of white-nose syndrome detection.
- Optimized methods support early fungal invasion detection and informed wildlife management decisions.
