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Diseases of Aquatic Organisms

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DAO 54:127-134 (2003)  -  doi:10.3354/dao054127

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

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

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