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Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA


Wilson, TK and Carson, J and Bowman, JP, Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA, Journal of Microbiological Methods, 51, (2) pp. 163-170. ISSN 0167-7012 (2002) [Refereed Article]

DOI: doi:10.1016/S0167-7012(02)00055-6


A simple, high-throughput, low-cost polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol that detects the presence of 4 fg of DNA from four bacterial fish pathogens Yersinia ruckeri, Tenacibaculum maritimum (formerly Flexibacter maritimus), Lactococcus garvieae and Aeromonas salmonicida was developed. DNA amplification was undertaken in a biphasic system with free and bound PCR that are achieved in the one NucleoLink tube. Solid-phase amplicons were detected using biotin labelled hybridization probes and visualised colourimetrically with streptavidin-alkaline phosphatase and p-nitrophenylphosphate as substrate. PCR and hybridization took less than 8 h to perform with maximum signal output for femtogram amounts of template DNA achieved within 24 h. Implementation and optimization of the protocol is discussed. © 2002 Elsevier Science B.V. All rights reserved.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Microbiology
Research Field:Bacteriology
Objective Division:Animal Production and Animal Primary Products
Objective Group:Fisheries - wild caught
Objective Field:Fisheries - wild caught not elsewhere classified
UTAS Author:Wilson, TK (Ms Teresa Wilson)
UTAS Author:Carson, J (Dr Jeremy Carson)
UTAS Author:Bowman, JP (Associate Professor John Bowman)
ID Code:24801
Year Published:2002
Web of Science® Times Cited:14
Deposited By:Agricultural Science
Deposited On:2002-08-01
Last Modified:2011-11-08

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