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.
Wilson
T
T
Fish Health Unit, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Australia. Teresa.Wilson@dpiwe.tas.gov.au
Carson
J
J
Bowman
J
J
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