DAO 139:233-243 (2020)  -  DOI: https://doi.org/10.3354/dao03482

ABSTRACT: Accurate detection of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is critical for wildlife disease research; however, false negatives in detection do occur. Here we compared different DNA extraction methods to determine the threshold for Bd detection and identify an optimal extraction method to improve detection and quantification of the pathogen. We extracted both lab-created cell suspension standards using PrepMan Ultra, Chelex resin, and 3 spin column DNA extraction kits (Qiagen DNeasy Blood and Tissue, Zymo Quick DNA miniprep, and IBI gMAX mini kit), and further compared extraction methods using field-collected samples. We found that when extracting Bd DNA from cells in lab-created culture, the spin column extraction methods and PrepMan Ultra were equivalent, while the resin method detected higher Bd DNA quantities, especially at higher loads. However, when swabs from live animals were analyzed, low Bd quantities were more than twice as likely to be detected using a spin column extraction than with the PrepMan Ultra extraction method. All tested spin column extraction methods performed similarly across both field and lab samples. Samples containing low Bd quantities yielded inconsistent detection and quantification of Bd DNA copies regardless of extraction method. To manage imperfect detection of Bd, we suggest that presence/absence analyses are more informative than attempting to quantify Bd DNA when quantities are low. Overall, we recommend that a cost-benefit analysis of target species susceptibility and epidemiology be taken into consideration when designing an experiment to determine the most appropriate DNA extraction method to be used, because sometimes detecting low Bd quantities is imperative to the study, whereas in other situations, detecting low DNA quantities is less important.

KEY WORDS: Chytridiomycosis · Amphibian disease · Pathogen detection · Detection threshold · False negative · DNA extraction · Qiagen DNeasy Blood and Tissue · PrepMan Ultra · Chelex resin