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 1 , E. Tobias Krause 2 , Molly C. Bletz 1 , An Martel 3 , Frank Pasmans 3 , Sebastian Steinfartz 1 and Miguel Vences 1
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  • 1 Zoological Institute, 38106 Braunschweig, Germany
  • 2 Friedrich-Loeffler-Institute, 29223 Celle, Germany
  • 3 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, 9820 Merelbeke, Belgium
Online Publication Date:
01 Jan 2019
Volume/Issue:
Volume 40: Issue 1
Article Type:
Research Article
DOI:
https://doi.org/10.1163/15685381-20181011
Keywords:
Batrachochytrium dendrobatidis; B. salamandrivorans; chytridiomycosis; epidemiology; pathogen survey; Prepman; Qiagen
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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.

In:
Amphibia-Reptilia
E-ISSN:
1568-5381
Print ISSN:
0173-5373
Publisher:
Brill
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Figures
  • View in gallery

    Experimental set up for pooled extraction experiment (experiment 2). Swabs were grouped in four groups with one to four swabs (pool size). One swab per group was inoculated with a pre-determined amount of chytrid zoospores (load) with the exception of the control on which no zoospores were added. This design was implemented for Batrachochytrium dendrobatidis and B. salamandrivorans. Load in zoospores per swab.

  • View in gallery

    Detectability and zoospore amounts of the detected loads for Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) according to the extraction method (PrepMan vs. Qiagen for Bsal), pool size, and load of the inoculated swab. O/E: Number of swabs with positive signal for the chytrid fungi and the total number of swabs processed. GE: Genomic equivalents, approximate number of zoospores per swab. SE: Standard error. Bold values indicate groups in which not all samples amplified.

  • View in gallery

    Costs (left) and time (right) necessary to process samples discriminated in x-axis. Grey-scale lines refer to the size of the pool with Qiagen extraction. Costs are shown in Euros, time is in hours.

Experimental set up for pooled extraction experiment (experiment 2). Swabs were grouped in four groups with one to four swabs (pool size). One swab per group was inoculated with a pre-determined amount of chytrid zoospores (load) with the exception of the control on which no zoospores were added. This design was implemented for Batrachochytrium dendrobatidis and B. salamandrivorans. Load in zoospores per swab.

Detectability and zoospore amounts of the detected loads for Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) according to the extraction method (PrepMan vs. Qiagen for Bsal), pool size, and load of the inoculated swab. O/E: Number of swabs with positive signal for the chytrid fungi and the total number of swabs processed. GE: Genomic equivalents, approximate number of zoospores per swab. SE: Standard error. Bold values indicate groups in which not all samples amplified.

Costs (left) and time (right) necessary to process samples discriminated in x-axis. Grey-scale lines refer to the size of the pool with Qiagen extraction. Costs are shown in Euros, time is in hours.
Index Card
Page Count:
29–39
Subjects:
Biology & Environmental Sciences, Biology
Copyright:
Copyright 2019 by Koninklijke Brill NV, Leiden, The Netherlands 2019
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