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DAO 54:127-134 (2003)
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Abstract
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Development of sensitive, high-throughput one-tube RT PCR enzyme hybridisation assay to detect selected bacterial fish pathogens
T. Wilson1,2,*, J. Carson1
1Fish Health Unit, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Tasmania 7249, Australia
2Fish Health Unit, Department of Primary Industries, Water and Environment, PO Box 46, Kings Meadows, Launceston, Tasmania 7249, Australia
*Email: teresa.wilson@dpiwe.tas.gov.au
ABSTRACT: Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease. To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of
ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed. The system uses
NucleoLinkTM strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 µl sample volume from selective
enrichment culture media. The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p nitrophenylphosphate.
KEY WORDS: RT-PCR-EHA · RT PCR ELISA · NucleoLink · High-throughput · Hybridisation
Full text in pdf format
Published in DAO Vol.
54, No. 2
(2003) on March 31
Print ISSN: 0177-5103; Online ISSN: 1616-1580.
Copyright © Inter-Research, Oldendorf/Luhe, 2003
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