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

conference contribution
posted on 2023-05-24, 14:23 authored by Andrew BridleAndrew Bridle, Tam, K, Crosbie, P, Barbara NowakBarbara Nowak

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.

History

Publication title

Proceedings of the Seventh International Symposium on Aquatic Animal Health

Pagination

58

Department/School

Institute for Marine and Antarctic Studies

Event title

Seventh International Symposium on Aquatic Animal Health

Event Venue

Portland, Oregon, USA

Date of Event (Start Date)

2015-08-31

Date of Event (End Date)

2015-09-04

Repository Status

  • Restricted

Socio-economic Objectives

Fisheries - aquaculture not elsewhere classified

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