title: Improved templated fluorogenic probes enhance the analysis of closely related pathogenic bacteria by microscopy and flow cytometry.
abstract: Templated fluorescence activation has recently emerged as a promising molecular approach to detect and differentiate nucleic acid sequences in vitro and in cells. Here, we describe the application of a reductive quencher release strategy to the taxonomic analysis of Gram-negative bacteria by targeting a single nucleotide difference in their 16S rRNA in a two-color assay. For this purpose, it was necessary to develop a release linker containing a quencher suitable for red and near-infrared fluorophores, and to improve methods for the delivery of probes into cells. A cyanine-dye labeled oligonucleotide probe containing the new quencher-release linker showed unprecedentedly low background signal and high fluorescence turn-on ratios. The combination of a fluorescein-containing and a near-IR emitting probe discriminated E. coli from S. enterica despite nearly identical ribosomal target sequences. Two-color analysis by microscopy and the first successful discrimination of bacteria by two-color flow cytometry with templated reactive probes are described.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, United States.
authors.forename: Raphael M
authors.initials: RM
authors.lastname: Franzini
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000145:classification / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019779:Salmonella enterica / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D019793:Fluorescein