Phages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a "proof of concept" bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples. This approach reduces testing time and enhances sensitivity to identify pathogens compared to traditional overnight plaque assay. Further, the assay has the ability to discriminate between live and dead cells since phages require live host cells to infect and replicate. To demonstrate its utility, phage MS2 amplification-coupled, bead-based sandwich type immunoassay on the Luminex® MAGPIX instrument for Escherichia coli detection was performed. The assay not only showed live cell discrimination ability but also a limit of E. coli detection of 1 × 102 cells/mL of live cells after a 3-h incubation. In addition, the sensitivity of the assay was not impaired in the presence of dead cells. These results demonstrate that bacteriophage amplification-coupled assay can be a rapid live cell detection assay compared to traditional culture methods and a promising tool for quick validation of bacterial inactivation. Combined with the unique multiplex bead chemistry of the Luminex® MAGPIX platform, the phage assay can be expanded to be an ultra-deep multiplex assay for the simultaneous detection of multiple pathogens using specific phages directed against the target pathogens.
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