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Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi

In: Amphibia-Reptilia
Authors:
Joana Sabino-Pinto Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany

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E. Tobias Krause Friedrich-Loeffler-Institute, Institute of Animal Welfare and Animal Husbandry, 29223 Celle, Germany

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Molly C. Bletz Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany

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An Martel Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium

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Frank Pasmans Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium

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Sebastian Steinfartz Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany

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Miguel Vences Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany

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Abstract

Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.

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