whole_WilsonTeresaKaye2003_thesis.pdf (11.55 MB)
Development of a streamlined, selective-enrichment culture, one-tube (RT-)PCR-enzyme hybridization assay to detect bacterial fish pathogens in covertly infected farmed salmonids
thesisposted on 2023-05-27, 15:28 authored by Wilson, TK
A new system which offers a low-cost and high performance means for detecting four bacterial pathogens Aeromonas salmonicida, Tenacibaculum maritimum, Lactococcus garvieae and Yersinia ruckeri in covertly infected farmed salmonid fish has been developed. The system couples Selective Enrichment Culture (SEC), Polymerase Chain Reaction (PCR) and Enzyme Hybridization Assay (EHA) technologies to provide streamlined high-throughput sample processing suitable for large scale surveillance and monitoring programs. The SEC media used for the technology were those developed by T.Wilson and J.Carson for the Cooperative Research Centre (CRC) for Aquaculture Ltd, except for the A. salmonicida medium which was developed using information obtained during the CRC project and by performing Minimum Inhibitory Concentration (MIG) assays with 32 antimicrobial agents that were not previously tested. Laboratory sensitivity of the A. salmonicida medium was 100% as determined by Most Probable Number (MPN) analysis and specificity of the medium was 97%. Bacterial DNA and RNA were extracted from the SEC media using guanidinium isothiocyanate (GuSCN) and Whatman Polyfiltronics GF/B 96-well Uni-filter plates. Sensitivity of the system as determined by PCR was between 1 and 16 CFU per 200 ˜í¬¿I of selective-enrichment medium for DNA and between 1 and 9 CFU per 200 ˜í¬¿I of selective-enrichment medium for RNA. The use of vacuum filtration and the 96-well glass microfibre filter plate allowed for rapid high-throughput and inexpensive extraction. Due to the high sensitivity of PCR, the technology is prone to false-positive results due to amplicon carry-over. To help prevent the occurrence of these erroneous results the photochemical IP-10 was added to the procedure. IP-10 inactivates PCR amplicons rendering them incapable of re-amplification in subsequent PCR reactions. Nucleolink ‚Äövë¬¢ PCR-enzyme hybridization strips were used to perform sensitive streamlined (RT)PCR-EHA. For each bacterium 4 fg of pure target DNA or RNA was detected. With this system only one tube per sample from cDNA to EHA was required, decreasing the cost and time involved in sample transfer and decreasing the risk of cross-contamination between samples. In laboratory trials the final SEC-(RT-)PCR-EHA system proved to be very sensitive with 1-16 CFU from selective-enrichment culture detected. Specificity of the system was >99%. The system was also rapid with the 96-well nucleic acid extraction to EHA results achieved in as little as 8 hours. Test validation with field samples was achieved by determining test specificity and sensitivity from an epidemiological perspective. During this testing the sensitivity and specificity of the system matched that obtained using purified nucleic acids in the laboratory. Validation was conducted using a total of 10 fish trials using fish from different environments and from different sample sources. The system described here uses two types of technology, PCR and RT-PCR. The SEC-PCR-EHA system detects genomic DNA from the target pathogen, demonstrating evidence of infection, past or present and is ideal for use in surveillance programs and for quarantine. The SEC-RT-PCR-EHA system detects ribosomal RNA from the target pathogens. This system gives a more accurate indication of the presence of live bacteria and therefore of live covert infection and is useful when monitoring changes in the disease status of a population of fish over a short period of time. The system was developed using inexpensive materials instead of proprietary products, and disposable equipment usage was minimised in an effort to keep the system low-cost. The sensitivity of the system was maximised to ensure the detection of covertly infected fish and specificity of the system was as good as the PCR primers would allow. The system utilises 96-well technology to minimise the volume of reagents and to enable streamlined sample processing using a multichannel pipette. In summary, a low-cost, high-performance and streamlined highthroughput sampling system has been developed.
Rights statementCopyright 2003 the author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s). Thesis (Ph.D)--University of Tasmania, 2003. Includes bibliographical references