Utilization of fast qPCR techniques to detect the amphibian chytrid fungus: a cheaper and more efficient alternative method

A pathogen of great significance to amphibian populations is the chytrid fungus, Batrachochytrium dendrobatidis (Bd). It has been demonstrated as causing recent epizootics in wild populations and is also widely found in captive animals. It is listed as a notifiable disease within the pet, bait and food trade because of its risk of introduction into wild populations. Due to this status, there has been much emphasis on reliably identifying and quantifying the pathogens in amphibians. Quantitative polymerase chain reaction (qPCR) has served as the recent standard for identifying this pathogen's presence. Newer technologies have greatly improved these reactions enabling researchers to use smaller volumes and run the reactions in less time. These fast' qPCR chemistries are gaining popularity because the reduced volumes required to run the reactions can save funding resources and reduce the time to data acquisition. In this study, we compare the results from differing reaction methodologies using the same DNA extracts from pathogens collected from wild sampled amphibians. In addition to comparing the standard methodology and fast methodology for both pathogens, we also conducted a reduced volume methodology using the standard TaqMan chemistry for Bd. Estimated pathogen loads from 114 field swab samples were compared among methodologies. We found that for Bd, all three methodologies produced similar results for prevalence (presence/absence) estimates. In terms of estimating pathogen loads in the samples, both the standard and fast methodologies produced comparable estimates but the reduced volume methodology exhibited significantly lower values. Therefore, it appears that the fast methodology is adequate for use with Bd, and potentially several other wildlife pathogens, in estimating both prevalence and quantity, but the reduced volume methodology is inadequate and not recommended for use in quantifying samples.