title: Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout.
abstract: Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-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-β-D-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo(®)) β-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-μm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °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 β-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.
authors: Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors: Sean
authors: S
authors: Burnham
authors: Jing
authors: J
authors: Hu
authors: Hany
authors: H
authors: Anany
authors: Lubov
authors: L
authors: Brovko
authors: Frederique
authors: F
authors: Deiss
authors: Ratmir
authors: R
authors: Derda
authors: Mansel W
authors: MW
authors: Griffiths
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics / Q000502:physiology*; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000821:virology*; D005618:Fresh Water / Q000737:chemistry / Q000382:microbiology*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*