Combined capillary electrophoresis and DNA-fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture.

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain-forming cells, where differences in cell size, shape, or chain length may lead to broad, "unfocusable" distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell-specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence-based labeling of whole target cells. In this work, we combined a simple CE-based presence/absence test with FISH to develop a bacterial detection assay having an additional "layer" of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10-15 min), bacterial populations were highly focused ( approximately 2-3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of approximately 300 injected E. coli cells, suggesting the possibility for single-cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE-FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.