Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout
Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout
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DOI：
10.1007/s00216-014-7985-3
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发表时间：
2014-09-01
影响因子：
4.3
通讯作者：
Griffiths, Mansel W.
中科院分区：
文献类型：
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作者：
Burnham, Sean;Hu, Jing;Griffiths, Mansel W.
使用野生型 T4 噬菌体和携带 β-半乳糖苷酶基因的重组报告 lac Z T4 噬菌体，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。该反应在纸基便携式培养装置上进行，以限制试剂的扩散，从而提高测定的灵敏度，并避免处理大量样品，使该测定适合现场分析。该测定中测试了显色（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(A(R))）β-半乳糖苷酶底物。水样首先通过0.45μm孔径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基装置中，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、光度计或发光成像装置记录信号（颜色、发光）的发展。结果表明，当野生型 T4 噬菌体或重组 lacZ T4 噬菌体分别用于测定时，可在 8 小时内目视检测到低至 40 或 < 10 菌落形成单位 (cfu) ml(-1) 的大肠杆菌。应用生物发光β-半乳糖苷酶底物可以在5.5小时内可靠地检测<10 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测定的特异性。快速大肠杆菌测定的方案包括过滤水样、在纸基培养装置中的过滤器上短暂孵育、添加噬菌体和β-半乳糖苷酶底物以及记录/处理累积的颜色或发光信号。
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.