BSA reduces inhibition in a TaqMan® assay for the detection of Batrachochytrium dendrobatidis

Garland, SM; Baker, Anthony L.; Phillott, Andrea D.; Skerratt, Lee F.

doi:10.3354/dao02053

Cited by 72 publications

(73 citation statements)

References 3 publications

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“…DNA was extracted from the swabs using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia, CA), with a final elution volume of 200 lL. Extracted DNA was then analyzed using quantitative, realtime PCR (qPCR, 7500 system, Applied Biosystems, Carlsbad, CA) following Boyle et al (2004) with the following minor modifications: each sample was diluted 1:10 with doubledeionized water and 0.7 lL of bovine serum albumin (BSA, Applied Biosystems, Carlsbad, CA) was added to each reaction well prior to amplification (following Garland et al 2010). We ran each sample in triplicate, with a positive and negative control, and a series of dilution standards.…”

Section: Bd Assaysmentioning

confidence: 99%

Fitness Consequences of Infection by Batrachochytrium dendrobatidis in Northern Leopard Frogs (Lithobates pipiens)

et al. 2013

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The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been linked to amphibian declines and extinctions worldwide. The pathogen has been found on amphibians throughout eastern North America, but has not been associated with mass die-offs in this region. In this study, we conducted laboratory experiments on the effects of Bd infection in a putative carrier species, Lithobates pipiens, using two estimators of fitness: jumping performance and testes morphology. Over the 8-week study period, peak acceleration during jumping was not significantly different between infected and uninfected animals. Peak velocity, however, was significantly lower for infected animals after 8 weeks. Two measures of sperm production, germinal epithelium depth, and maximum spermatic cyst diameter, showed no difference between infected and uninfected animals. The width, but not length, of testes of infected animals was significantly greater than in uninfected animals. This study is the first to show effects on whole-organism performance of Bd infection in post-metamorphic amphibians, and may have important long-term, evolutionary implications for amphibian populations co-existing with Bd infection.

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Section: Bd Assaysmentioning

confidence: 99%

Fitness Consequences of Infection by Batrachochytrium dendrobatidis in Northern Leopard Frogs (Lithobates pipiens)

et al. 2013

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“…This master cocktail was split into 2 tubes, one of which received TaqMan exogenous internal positive control (IPC; Applied Biosystems, Part No. 43083283) following Garland et al (2010). The other tube was left as 'normal' master mix.…”

Section: Real-time Taqman Pcr Assaymentioning

confidence: 99%

“…Samples were run in duplicate using an Applied Biosystems 7900HT sequence detection system and a modified version of the protocol developed by Boyle et al (2004) and Garland et al (2010). To reduce costs, we used 20 µl (rather than 25 µl) reactions containing 10 µl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Part No.…”

Section: Real-time Taqman Pcr Assaymentioning

confidence: 99%

Evaluation of a filtration-based method for detecting Batrachochytrium dendrobatidis in natural bodies of water

Hyman¹,

Collins²

2012

Dis. Aquat. Org.

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Infectious diseases are emerging as a significant threat to wildlife. The resulting increased effort to monitor wildlife diseases is driving the development of innovative pathogen monitoring techniques, including many polymerase chain reaction (PCR)-based diagnostics. Despite the utility of these PCR-based techniques, there is still much to be learned about their ability to accurately detect target pathogens in nature. We assessed the diagnostic sensitivity of a PCR-based water filtration technique to detect the directly transmitted aquatic fungal pathogen Batrachochytrium dendrobatidis (Bd) by comparing the results of 4 repeated filter sampling events from 20 ponds to those of skin swabs from ca. 60 boreal chorus frogs Pseudacris maculata from each pond. Filters failed to detect Bd in 31 to 77% of the swab-positive ponds, depending on the time of sampling. However, after 3 repeated sampling events, filtration of small volumes of water (ca. 600 ml) correctly identified 94% of the ponds that tested Bd positive with swabbing, with the highest rates of detection occurring after breeding but before larvae reached metamorphosis. Our results are a case study demonstrating the importance of timing and resampling for the detection of an aquatic microbial pathogen, Bd, from water. This will be a useful technique for monitoring Bd, but additional data are needed to test the degree to which our findings are species or population specific. Future studies need to examine the sensitivity of this technique in other habitats and species that host Bd. These studies will aid in the development of cost-effective monitoring regimes for Bd and potentially other aquatic pathogens.

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“…Presence of the pathogen will be detected using Bdspecific primers: Bd1a (5 0 -CAGTGTGCCATATGTCACG-3 0 ) and Bd2a (5 0 -CATGGTTCATATCTGTCCAG-3 0 ) (Annis , designed from the ITS1 and ITS2 regions, respectively. PCRs will be performed following Federici et al (2008), with the only exception of adding bovine serum albumin (BSA) to PCRs to reduce amplification inhibition for all DNA extracts (Garland et al 2010). PCR products will be loaded onto 2% agarose gels, stained with ethidium bromide, and observed under ultraviolet light (365 nm).…”

Section: Analysis Of Swab Samplesmentioning

confidence: 99%

Pre-emptive National Monitoring Plan for Detecting the Amphibian Chytrid Fungus in Madagascar

Weldon

Crottini²,

Bollen³

et al. 2013

EcoHealth

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BSA reduces inhibition in a TaqMan® assay for the detection of Batrachochytrium dendrobatidis

Cited by 72 publications

References 3 publications

Fitness Consequences of Infection by Batrachochytrium dendrobatidis in Northern Leopard Frogs (Lithobates pipiens)

Fitness Consequences of Infection by Batrachochytrium dendrobatidis in Northern Leopard Frogs (Lithobates pipiens)

Evaluation of a filtration-based method for detecting Batrachochytrium dendrobatidis in natural bodies of water

Pre-emptive National Monitoring Plan for Detecting the Amphibian Chytrid Fungus in Madagascar

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