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Open Access Methodology article

Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR

Nicole Strepparava12*, Thomas Wahli2, Helmut Segner2 and Orlando Petrini3

Author Affiliations

1 Laboratory of Applied Microbiology, University of Applied Sciences and Arts of Southern Switzerland, Via Mirasole 22a, 6500 Bellinzona, Switzerland

2 Centre for Fish and Wildlife Health, University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland

3 POLE Pharma Consulting, Breganzona, Switzerland

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BMC Microbiology 2014, 14:105  doi:10.1186/1471-2180-14-105

Published: 26 April 2014

Abstract

Background

Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed.

Results

We describe a qPCR technique based on the single copy gene β’ DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes.

Conclusions

This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.