Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens.
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 NucleoLink 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 microl 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.
Fish Health Unit, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Tasmania 7249, Australia. teresa.wilson@dpiwe.tas.gov.au
T
T
Wilson
J
J
Carson
D000333:Aeromonas / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D005399:Fishes / Q000382:microbiology*; D017045:Lactococcus / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D015007:Yersinia / Q000235:genetics / Q000302:isolation & purification
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S