A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested existence of two clades amphibian Perkinsea: putatively non-pathogenic clade linked asymptomatic infections tadpoles in Africa, Europe South America, pathogenic disease mass mortality events North America. Here, we describe development duplex TaqMan qPCR assay detect discriminate between rDNA sequences from both tissues. The uses single primer pair target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared clades, dual-labelled probes this fragment that is diagnostic for each clade. This enables rapid screening groups, allowing detection, primarily phylogenetic group associated with outbreaks, secondarily no current relationship identified. Incorporation our novel into routine surveillance populations will allow assessment incidence protist clade, thereby providing improved understanding infection pervasiveness method underpin future conservation planning.