Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system
Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system
复制标题
DOI：
10.1007/s00216-019-02095-4
复制
发表时间：
2019-11-01
影响因子：
4.3
通讯作者：
Talbert, Joey N.
中科院分区：
文献类型：
--
作者：
Singh, Sangita;Hinkley, Troy;Talbert, Joey N.
报告噬菌体系统已成为检测食品和水中细菌的一项有前景的技术。然而，这些测定的灵敏度通常受到表达报告基因的浓度以及与样品相关的基质干扰的限制。在这项研究中，噬菌体 T7 被设计为在感染大肠杆菌后过表达与碳水化合物结合模块 (ALP*-CBM) 融合的突变碱性磷酸酶，从而在模型系统中实现比色检测。采用磁性纤维素颗粒分离和浓缩细菌裂解液中过表达的 ALP*-CBM。用工程噬菌体感染大肠杆菌导致定量限制为 1.2 x 10(5) CFU，相当于使用比色测定和 100 mL 样品体积时，3.5 小时内 1.2 x 10(3) CFU/mL。当采用富集步骤时，可以在 8 小时内从 100 mL 样品体积中目视检测到 < 10(1) CFU/mL。这些结果表明，亲和标签修饰的酶与材料支持物结合可以提供一种简单有效的方法来提高基于噬菌体的测定的信号灵敏度。图形摘要
Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 x 10(5) CFU, equating to 1.2 x 10(3) CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 10(1) CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays.Graphical abstract