Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the beta-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of beta-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-beta-d-galactopyranoside, CPRG) and bioluminescent (6-O-beta-galactopyranosyl-luciferin, Beta-Glo(A (R))) beta-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-mu m pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 A degrees C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or < 10 colony-forming units (cfu) ml(-1) of E. coli can be detected visually within 8 h when wild-type T4 bacteriophage or recombinant lacZ T4 bacteriophage were used in the assay, respectively. Application of the bioluminescent beta-galactosidase substrate allowed reliable detection of < 10 cfu ml(-1) within 5.5 h. The specificity of the assay was demonstrated using a panel of microorganisms including Aeromonas hydrophila, Enterobacter cloacae, E. coli, and Salmonella Typhimurium. Scheme for rapid E. coli assay including filtration of water sample, short incubation on the filter in a paper-based culture device, addition of bacteriophage and [beta]-galactosidase substrate, and recording/processing of the accumulated color or luminescence signal.