Rapid, expression‐free bacteriophage‐based specific detection of target bacteria by conditional release of encapsidated reporter molecules

Abstract Rapid diagnosis of infectious diseases is of paramount importance to prevent or control outbreaks and pandemics. Detection of bacteria is commonly performed using culture‐based and molecular detection methods, which cannot address the need for quick, specific and cheap diagnostics. Bacteriophage‐based assays rely on the rapidity, specificity, contaminant‐tolerance and effectiveness of phage‐host interactions and can be engineered with fluorescence or luminescence‐based reporters. Previous attempts, however, required transcription and translation of reporter genes, leading to long assays and restrictive protocols. Here, we shortened the signal generation time by detecting the injection of a phage protein, thereby circumventing the need for gene expression altogether. In our model diagnostic assay, we demonstrate that injection of the N‐terminal fragment of the split nanoluciferase protein of Oplophorus gracilirostris, fused to the products of genes g6.7 or g14 of phage K1F, is detectable upon injection into an Escherichia coli cell as early as 3 min after phage addition. The engineered phages generate a signal upon exposure to cognate K1—but not to non‐cognate K5 capsule‐enclosed E. coli cells, confirming the specificity of our system. The early luminescent signal and the ability to detect as few as 10 4 bacteria may open the way to the development of a rapid diagnostic tool based on phage‐mediated protein injection.