School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou, China.
Mikrochim Acta. 2023 May 5;190(6):202. doi: 10.1007/s00604-023-05784-1.
A colorimetric detection method for Escherichia coli (E. coli) in water was established based on a T7 phage tail fiber protein-magnetic separation. Firstly, the tail fiber protein (TFP) was expressed and purified to specifically recognize E. coli, which was verified by using fusion protein GFP-tagged TFP (GFP-TFP) and fluorescence microscopy. Then TFP conjugated with magnetic beads were applied to capture and separate E. coli. The TFP was covalently immobilized on the surface of magnetic beads and captured E. coli as verified by scanning electron microscopy (SEM). Finally, polymyxin B was used to lyse E. coli in solution and the released intracellular β-galactosidase (β-gal) could hydrolyze the colorimetric substrate chlorophenol red-β-D-galactopyranoside (CPRG), causing color change from yellow to purple. The high capture efficiencies of E. coli ranged from 88.70% to 95.65% and E. coli could be detected at a concentration of 10 CFU/mL by naked eyes. The specificity of the chromogenic substrate was evaluated using five different pathogen strains as competitors and tests with four kinds of real water samples showed recoveries of 86.00% to 92.25%. The colorimetric changes determined by visual inspection can be developed as an efficient platform for point-of-care detection of E. coli in resource-limited regions.
建立了一种基于 T7 噬菌体尾纤维蛋白-磁分离的水中大肠杆菌（E. coli）比色检测方法。首先，表达和纯化了尾纤维蛋白（TFP），以特异性识别大肠杆菌，这通过融合蛋白 GFP 标记的 TFP（GFP-TFP）和荧光显微镜得到验证。然后，将与磁珠偶联的 TFP 用于捕获和分离大肠杆菌。通过扫描电子显微镜（SEM）验证了 TFP 被共价固定在磁珠表面并捕获了大肠杆菌。最后，多粘菌素 B 用于裂解溶液中的大肠杆菌，释放的胞内β-半乳糖苷酶（β-gal）可水解比色底物氯酚红-β-D-半乳糖苷（CPRG），导致颜色从黄色变为紫色。大肠杆菌的高捕获效率范围为 88.70%至 95.65%，并且可以通过肉眼检测到浓度为 10 CFU/mL 的大肠杆菌。使用五种不同的病原体菌株作为竞争者评估了比色底物的特异性，并且对四种实际水样的测试显示回收率为 86.00%至 92.25%。通过目视检查确定的比色变化可以开发为在资源有限地区进行即时检测大肠杆菌的有效平台。
