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

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  Electrophoresis. 2008 Jun;29(12):2477-84.


  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.

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