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

Autor: Brosnahan CL; Ministry for Primary Industries, Wellington, New Zealand; School of Veterinary Science, Massey University, Palmerston North, New Zealand. Electronic address: cara.brosnahan@mpi.govt.nz., Georgiades E; Ministry for Primary Industries, Wellington, New Zealand., McDonald C; Ministry for Primary Industries, Wellington, New Zealand., Keeling SE; Ministry for Primary Industries, Wellington, New Zealand., Munday JS; School of Veterinary Science, Massey University, Palmerston North, New Zealand., Jones B; Murdoch University, School of Veterinary and Life Sciences, Perth, WA, Australia.
Jazyk: angličtina
Zdroj: Journal of microbiological methods [J Microbiol Methods] 2019 Apr; Vol. 159, pp. 186-193. Date of Electronic Publication: 2019 Mar 12.
DOI: 10.1016/j.mimet.2019.03.008
Abstrakt: 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 10 3  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 10 6  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.
(Copyright © 2019 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: