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An optimized real-time PCR for the detection and quantification of viable Neoparamoeba perurans using propidium monoazide

Citation

Bridle, AR and Tam, K and Crosbie, P and Nowak, BF, An optimized real-time PCR for the detection and quantification of viable Neoparamoeba perurans using propidium monoazide, Proceedings of the Seventh International Symposium on Aquatic Animal Health, 31 August - 4 September 2015, Portland, Oregon, USA, pp. 58. (2015) [Conference Extract]


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Abstract

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. Despite recent advances by our research group, culturing this pathogen from sources other than the host remain problematic. Therefore current environmental detection methods rely on molecular techniques namely, polymerase chain reaction (PCR) and in situ hybridization (ISH). Previously we developed 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 DNA extraction technique and quantitative real-time PCR that reduces the chance of acquiring a false negative 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 experimental AGD infection tank and a variety of environmental sites including those used to culture Atlantic salmon (Salmo salar L.) in Tasmania, Australia. Amoebae were detected and quantified from sites in and closely surrounding cage culture of Atlantic salmon.

Item Details

Item Type:Conference Extract
Keywords:Neoparamoeba perurans, AGD, real-time PCR
Research Division:Agricultural and Veterinary Sciences
Research Group:Fisheries Sciences
Research Field:Aquaculture
Objective Division:Animal Production and Animal Primary Products
Objective Group:Fisheries - Aquaculture
Objective Field:Fisheries - Aquaculture not elsewhere classified
Author:Bridle, AR (Dr Andrew Bridle)
Author:Tam, K (Mr King Tam)
Author:Crosbie, P (Dr Philip Crosbie)
Author:Nowak, BF (Professor Barbara Nowak)
ID Code:104994
Year Published:2015
Deposited By:IMAS Research and Education Centre
Deposited On:2015-12-02
Last Modified:2016-01-27
Downloads:0

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