Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA.
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.
Fish Health Unit, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Australia. Teresa.Wilson@dpiwe.tas.gov.au
T
T
Wilson
J
J
Carson
J
J
Bowman
D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005399:Fishes / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial