Fast quantitative PCR, locked nucleic acid probes and reduced volume reactions are effective tools for detecting Batrachochytrium dendrobatidis DNA

Abstract

The fungal pathogen Batrachochytrium dendrobatidis threatens amphibian populations around the world. The ability to detect this pathogen on infected animals and in the environment is critical for understanding and controlling this pandemic. We tested several advances in quantitative PCR (qPCR) techniques to detect B. dendrobatidis DNA. We used a fast PCR thermocycler and enzymes that reduced the volume and the duration of the reaction. We also compared a conventional TaqMan minor groove binding (MGB) probe to an identical locked nucleic acid (LNA) counterpart. The fast qPCR reaction had a high degree of sensitivity to B. dendrobatidis DNA. The LNA probe was effective for detecting B. dendrobatidis DNA and produced results similar to those of the MGB probe. The modifications that we tested can improve the cost, time efficiency and specificity of quantitative PCR as a tool for detecting pathogen DNA. KEY WORDS: Batrachochytrium dendrobatidis · Quantitative PCR · Locked nucleic acids · Minor groove binding probe · Amphibian disease Resale or republication not permitted without written consent of the publisherDis Aquat Org 97: 249-253, 2012 250 cost, time efficiency, and specificity of the technique.Recent advances in qPCR have broadened the capability and accessibility of this approach to pathogen detection. They include faster polymerase enzymes, lower cost instrumentation with increased temperature ramp speeds and lower reaction volume requirements, and development of different types of fluorescent probes for detection of target sequences.First, we took advantage of new fast qPCR technology. The cost of fast quantitative machines is much lower than earlier instrument models, although the cost of fast reagents is slightly higher than their conventional counterparts. The greater detection sensitivity of the fast machines and the development of enzymes that can withstand faster temperature changes have reduced PCR reaction volumes and total cycle times from approximately 25 µl and 2 h, to 10 µl and 30 min, effectively lowering the actual cost per reaction. Yoder & Fishel (2008) showed that, in many cases, fast qPCR technology is compatible with established detection protocols. In the present study, we attempted to reduce the reaction volume and cycle times of 25 µl and 75 s used by Boyle et al. (2004) to 10 µl and 21 s.Finally, we tested different qPCR probe chemistries to assess the relative effectiveness and benefit of each. We compared the efficacy of the TaqMan (Applied Biosystems) minor groove binder (MGB) probe used by Boyle et al. (2004) to more recently developed locked nucleic acid (LNA) probe chemistry (Vester & Wengel 2004). Detection of genomic regions of interest requires sequence-specific primers that define the target area, while higher levels of specificity require the use of fluorescentlabeled probes that hybridize to a region between the primers and differentiate closely related sequences. The challenge of designing compatible primers and probes that satisfy reaction stringency requir...

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