File(s) not publicly available
Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA
journal contribution
posted on 2023-05-16, 13:43 authored by Wilson, TK, Carson, J, John BowmanJohn BowmanA 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. © 2002 Elsevier Science B.V. All rights reserved.
History
Publication title
Journal of Microbiological MethodsVolume
51Pagination
163-170ISSN
0167-7012Department/School
Institute for Marine and Antarctic StudiesPublisher
Elsevier Science BVPlace of publication
Amsterdam, NetherlandsRepository Status
- Restricted