Rapid and Specific Detection of <i>Escherichia coli</i> O157:H7 Using Engineered Phage Tailspike Protein-Based Magnetic Assisted Luminescence Assay

Escherichia coli O157:H7 is a highly virulent strain of E. coli that can cause food poisoning and infections such as hemolytic uremic syndrome, even in small quantities. To prevent and mitigate outbreaks, the rapid and sensitive detection of E. coli O157:H7 is crucial. Although antibodies have been widely used as biorecognition elements for bacterial detection, their high cost and cross-reactivity cause considerable frustration among researchers, calling for more cost-effective and specific bioprobes. Here, we engineered bacteriophage tail spike proteins (TSP) by eliminating active sites for strong binding and established a TSP-based magnetic-assisted luminescence assay (T-MALA) for the specific detection of E. coli O157:H7 cells. In the T-MALA, we utilized TSP-conjugated Dynabeads for capturing and nanoluciferase-fused TSP as a detection probe. After magnetic separation, the cell-bound Nluc-mTSPs generated luminescence upon exposure to its substrate, furimazine, enabling the detection of E. coli O157:H7 cells. After optimization, the entire process was completed in less than 1 h, with a low experimental detection limit (LOD) of 20 CFU/mL, and showed high specificity for E. coli O157:H7 strains. This assay exhibited a robust linear correlation between the luminescence values and bacterial concentrations ranging from 30–10 5 CFU/mL. Furthermore, T-MALA was able to detect E. coli O157:H7 cells in various food matrices, such as milk, lettuce, and ground beef, which are commonly associated with E. coli O157:H7 contamination. These results demonstrate the potential of T-MALA as an alternative strategy for rapid detection of E. coli O157:H7.