Journal article

Optimized Batrachochytrium dendrobatidis DNA extraction of swab samples results in imperfect detection particularly when infection intensities are low.

Laura A Brannelly, Daniel P Wetzel, Matt West, Corinne L Richards-Zawacki

Diseases of Aquatic Organisms | Inter Research | Published : 2020

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 cell..

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Grants

Awarded by Australian Research Council


Awarded by US National Science Foundation (IOS: Project)


Awarded by US Department of Defense (SERDP: Project)


Funding Acknowledgements

We thank Michel Ohmer, Lydia Zimmerman, Trina Wantman, Jakub Zegar, and Phoebe Reuben with help collecting the field samples; Caitlin Nordheim for help analyzing the samples; and Veronica Saenz, Jamie Voyles, Kimberly Chen, Lee Berger, and Mary Toothman for providing and maintaining the Bd strains. We thank Chris Davis, Jessica Barabas, Abdul Jabbar, and the Pymatuning Laboratory of Ecology for providing the lab space to conduct some of the molecular work. The work was funded through the Australian Research Council Discovery for Early Career Research Award scheme (DE180101395), the University of Melbourne's Faculty of Veterinary and Agricultural Sciences, University of Pittsburgh's Dietrich School of Arts and Sciences, the US National Science Foundation (IOS: Project No. 1649443), and the US Department of Defense (SERDP: Project No. RC-2638). This research was conducted under the University of Pittsburgh Institutional Animal Care and Use Committee protocol 16027711. Animal surveys were conducted with permission from the Pennsylvania Fish and Boat Commission (permit 2017-01-0177), and from the Louisiana Department of Wildlife and Fisheries (permit LNHP-17-029).