Department of Food Science, Cornell University, Ithaca, NY, 14853, United States.
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, 50011, United States.
Sci Rep. 2018 Oct 2;8(1):14630. doi: 10.1038/s41598-018-33097-4.
Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.
美国饮用水标准规定，每 100 毫升水中不得含有任何普通大肠杆菌。饮用水中存在大肠杆菌表明存在有利于病原体污染的环境条件。因此，快速、特异性地对受污染饮用水中的大肠杆菌进行计数，对于减轻对公众健康的重大风险至关重要。为了应对这一挑战，我们开发了一种基于噬菌体的膜过滤检测方法，该方法采用新型融合报告酶，在不到传统富集检测所需时间一半的情况下，对大肠杆菌进行完全定量。由于其信号强、体积小、背景低，我们选择了荧光素酶和碱性磷酸酶作为报告探针，这两种酶都是专门为提高酶活性而设计的。报告酶的基因与特定于纤维素的碳水化合物结合模块的基因融合。然后，这些构建体被插入到专门感染被困在纤维素过滤器上的大肠杆菌的大肠杆菌特异性噬菌体 T7 中。在感染过程中，报告酶在裂解感染周期后从细菌细胞中表达和释放。结合模块促进了报告探针在靠近裂解细胞的纤维素过滤器上的固定化。加入底物后，可通过肉眼观察或分别使用碱性磷酸酶和荧光素酶报告基因的生物发光成像来确定大肠杆菌细胞的位置和数量。结果，开发了一种能够定量检测饮用水中大肠杆菌的检测方法，其结果与现有方法相似，但检测时间不到一半。
