Bacteriophages represent rapid, readily targeted, and easily produced
molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific
protease, which is expressed during
infection of the target cell. Upon lysis, the
protease is released to cleave a reporter
peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (
TEV) protease; (ii) expression of
TEV protease by Escherichia coli following
infection by our modified T7, an average of 2000 units of
protease per phage are produced during
infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via
TEV protease activity using a fluorescent
peptide and using a designed target
peptide for matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-
protease-
peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.