Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples

C.L. Brosnahan; E. Georgiades; C. McDonald; S.E. Keeling; J.S. Munday; B. Jones

doi:10.1016/j.mimet.2019.03.008

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Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples

Journal article

Open access

Peer reviewed

Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples

C.L. Brosnahan, E. Georgiades, C. McDonald, S.E. Keeling, J.S. Munday and B. Jones

Journal of Microbiological Methods, Vol.159, pp.186-193

2019

DOI: https://doi.org/10.1016/j.mimet.2019.03.008

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Abstract

A PCR protocol was optimised and validated for the detection of viable Tenacibaculum maritimum cells in salmon skin tissue. Viability conventional (vPCR) and quantitative PCR (v-qPCR) assays both had a limit of detection of 103 CFU mL−1 viable cells. The v-qPCR assay showed a linear quantification over 4 log units. Conventional vPCR showed complete signal suppression when only dead cells were present at concentrations lower than 106 CFU mL−1. While the v-qPCR did not result in complete suppression when only dead cells were present, a method was developed to determine if viable cells were present based on the % Δ in cycle threshold (Ct) value. The procedure was validated for high-throughput processing and an enrichment protocol was validated to reliably detect low concentrations of viable cells both with and without a high background of dead cells. Performing this protocol on naturally infected tissues showed that vPCR and v-qPCR reduced the potential for false positives compared to using conventional PCR and qPCR. The optimised protocol developed for this study provides an efficient, reliable and robust alternative for the detection of viable T. maritimum in skin tissue.

Details

Title: Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples
Authors/Creators: C.L. Brosnahan (Author/Creator) - Ministry for Primary Industries
E. Georgiades (Author/Creator) - Ministry for Primary Industries
C. McDonald (Author/Creator) - Ministry for Primary Industries
S.E. Keeling (Author/Creator) - Ministry for Primary Industries
J.S. Munday (Author/Creator) - Massey University
B. Jones (Author/Creator)
Publication Details: Journal of Microbiological Methods, Vol.159, pp.186-193
Publisher: Elsevier BV
Identifiers: 991005544327607891
Copyright: © 2019 Elsevier B.V.
Murdoch Affiliation: School of Veterinary and Life Sciences
Language: English
Resource Type: Journal article

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Collaboration types: Domestic collaboration; International collaboration
Citation topics: 1 Clinical & Life Sciences; 1.42 Bacteriology; 1.42.853 Aquatic Pathogens
Web Of Science research areas: Biochemical Research Methods; Microbiology
ESI research areas: Microbiology