Burnham Sean, Hu Jing, Anany Hany, Brovko Lubov, Deiss Frederique, Derda Ratmir, Griffiths Mansel W
Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Anal Bioanal Chem. 2014 Sep;406(23):5685-93. doi: 10.1007/s00216-014-7985-3. Epub 2014 Jun 27.
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.
野生型T4噬菌体和携带β-半乳糖苷酶基因的重组报告lacZ T4噬菌体用于通过监测噬菌体介导的细胞裂解时β-半乳糖苷酶的释放来检测普通大肠杆菌。该反应在纸质便携式培养装置上进行，以限制试剂的扩散，从而提高检测的灵敏度，并避免处理大量样品，使该检测适用于现场分析。在检测中测试了显色（氯酚红-β-D-吡喃半乳糖苷，CPRG）和生物发光（6-O-β-吡喃半乳糖基-荧光素，Beta-Glo(®)）β-半乳糖苷酶底物。水样首先通过孔径为0.45μm的过滤器过滤以浓缩细菌。然后将过滤器放入含有营养培养基的纸质装置中，并在37°C下孵育4小时。将带有相应指示底物的噬菌体添加到装置中，并用数码相机、发光计或发光成像装置记录信号（颜色、发光）的发展。结果表明，当分别使用野生型T4噬菌体或重组lacZ T4噬菌体进行检测时，在8小时内可目视检测到低至40或<10个大肠杆菌菌落形成单位（cfu）ml(-1)。使用包括嗜水气单胞菌、阴沟肠杆菌、大肠杆菌和鼠伤寒沙门氏菌在内的一组微生物证明了该检测的特异性。
