Data from: DNA extraction method affects the detection of a fungal pathogen in formalin-fixed specimens using qPCR

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis

DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis

Adams_etal_2015_DNA_extraction_method_qPCR

The file contains all data used in the statistical analyses described in the materials and methods section of the paper. The dataset includes: “ZE” (number of zoospore equivalents detected for each treatment or swab event), “mass” (weight in grams of individual animals prior to formalin fixation), “frogid” (unique identifiers for each individual frog), and “SF” (success or failure of Batrachochytrium dendrobatidis detection for each swab or treatment type; 1= success, 0 = fail)

Comparison of overall Bd recovery success of Macherey-Nagel DNA FFPE extraction (MN) vs. PrepMan (PM).

<p>MN-extracted swabs were 31% more effective than PM at detecting Bd from formalin-fixed specimens that had been previously identified as Bd-positive in the field.</p

Post-fixation Bd detection success (top) and zoospore equivalents (bottom) across all swab events (n = 58).

<p>(a) Bd detection success was significantly greater for swab event E as compared to all other swab events (p < 0.0001). (b) The two ZE outliers in Event E are from specimens that had pre-preservation Bd loads of less than 1 zoospore equivalent and were not detected, in any number of runs, by any of the PM-extracted swabs. Results are based on analysis of singlicate data.</p

Probability of <i>Batrachochytrium dendrobatidis</i> (Bd) detection after formalin fixation.

<p>We used parameters from the best-fit generalized linear mixed models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135389#pone.0135389.t003" target="_blank">Table 3</a>) to predict Bd detection probabilities of the two different extraction methods, Macherey-Nagel DNA FFPE (MN), and PrepMan (PM). The differences in Bd detection probability between MN and PM are greatest at low and moderate Bd loads (as much as 50% at the median infection level of this study) and become more similar at higher Bd loads.</p

Treatments, replicate numbers, and optimizations for each specimen swabbing event, chronological from left to right.

<p>PM = PrepMan; MN = Macherey-Nagel DNA FFPE; GR = Genereleaser</p><p>^an = 62;</p><p>^bn = 47;</p><p>^cn = 50</p><p>^dn = 48</p><p>Treatments, replicate numbers, and optimizations for each specimen swabbing event, chronological from left to right.</p

Presence of <i>Batrachochytrium dendrobatidis</i> (<i>Bd</i>) detected on amphibians and vegetation sampled in Cusuco National Park, Honduras.

<p>Survey sites include Rio Cortecito (CO), Rio Danto (DA), and Rio Cusuco (CU). Average zoospore equivalent (ZSE) per qPCR reaction is reflected for all <i>Bd</i>-positive samples. Asterisk denotes the single sample that produced a positive reaction in 2/6 wells; all other samples produced <i>Bd</i>-positive reactions in 2/3, 3/3, or 3/6 wells.</p><p>Presence of <i>Batrachochytrium dendrobatidis</i> (<i>Bd</i>) detected on amphibians and vegetation sampled in Cusuco National Park, Honduras.</p

Recently metamorphosed <i>Plectrohyla dasypus</i> on terrestrial vegetation in Cusuco National Park, Honduras.

<p>(A) Amphibian as encountered on vegetation. (B) <i>Bd</i>-positive residue remaining on the leaf after amphibian removal.</p