Byron Brehm-Stecher - Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria.

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  Appl Environ   Microbiol. 2005 Sep;71(9):5451-7.



    Six fluorescein-labeled peptide nucleic acid oligomers     targeting Listeria-specific sequences on the 16S ribosomal     subunit were evaluated for their abilities to hybridize to     whole cells by fluorescence in situ hybridization (FISH). Four     of these probes yielded weak or no fluorescent signals after     hybridization and were not investigated further. The remaining     two FISH-compatible probes, LisUn-3 and LisUn-11, were     evaluated for their reactivities against 22 Listeria strains     and 17 other bacterial strains belonging to 10 closely related     genera. Hybridization with BacUni-1, a domain-specific     eubacterial probe, was used as a positive control for target     accessibility in both Listeria spp. and nontarget cells. RNase     T1 treatment of select cell types was used to confirm that     positive fluorescence responses were rRNA dependent and to     examine the extent of nonspecific staining of nontarget cells.     Both LisUn-3 and LisUn-11 yielded rapid, bright, and     genus-specific hybridizations at probe concentrations of     approximately 100 pmol ml(-1). LisUn-11 was the brightest probe     and stained all six Listeria species. LisUn-3 hybridized with     all Listeria spp. except for L. grayi, for which it had two     mismatched bases. A simple ethanolic fixation yielded superior     results with Listeria spp. compared to fixation in 10% buffered     formalin and was applicable to all cell types studied. This     study highlights the advantages of peptide nucleic acid probes     for FISH-based detection of gram-positive bacteria and provides     new tools for the rapid detection of Listeria spp. These probes     may be useful for the routine monitoring of food production     environments in support of efforts to control L. monocytogenes.  

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