AbstractPhages 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 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 forEscherichia colidetection was performed. The assay not only showed live cell discrimination ability but also a limit ofE. colidetection of 1×102cells/mL of live cells after a 3-hour 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 afforded by 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.
Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells
