| Summary: | The protozoan parasite Neoparamoeba perurans is the causative agent of amoebic gill disease(AGD) and an emerging threat to the aquaculture of marine finfish species worldwide. Despiterecent advances by our research group, culturing this pathogen from sources other than the hostremain problematic. Therefore current environmental detection methods rely on moleculartechniques namely, polymerase chain reaction (PCR) and in situ hybridization (ISH). Previously wedeveloped a qPCR assay able to detect a single 18S rRNA gene copy and readily detect a single N.perurans trophozoite filtered from a volume of water. Herein, we describe an optimized DNAextraction technique and quantitative real-time PCR that reduces the chance of acquiring a falsenegative from water samples containing large amounts of naturally occurring PCR inhibitors.Furthermore, we describe a modification of the assay allowing the discrimination of viable N.perurans by real-time PCR using propidium monoazide. The optimized qPCR method was applied to seawater samples collected from both an experimentalAGD infection tank and a variety of environmental sites including those used to culture Atlanticsalmon ( Salmo salar L.) in Tasmania, Australia. Amoebae were detected and quantified from sitesin and closely surrounding cage culture of Atlantic salmon.
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