Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like rana viruses. A synthetic genome template (gBlock (R)) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.