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

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 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 that work against uptake by untrained users. In this proof-of-concept work, we tested whether the signal generation time could be shortened by detecting the injection of a phage protein, thereby circumventing the need for gene expression altogether. 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 harbouring the C-terminal fragment, as early as 5 min 45 s after phage addition. We also demonstrate that the engineered phages generate a signal upon exposure to cognate K1 - but not to non-cognate K5 capsule-enclosed E. coli cells - indicating the specificity of our system.