Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r² > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction.