bc2003567_si_001.pdf (2.87 MB)
Improved Templated Fluorogenic Probes Enhance the Analysis of Closely Related Pathogenic Bacteria by Microscopy and Flow Cytometry
journal contribution
posted on 2011-09-21, 00:00 authored by Raphael M. Franzini, Eric T. KoolTemplated 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.
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templated reactive probesoligonucleotide probeTemplated Fluorogenic Probes Enhancenucleotide differencebackground signalbacteriaflow cytometryacid sequencestarget sequencesreductive quencher release strategyFlow CytometryTemplated fluorescence activationtaxonomic analysisRelated Pathogenic Bacteriarelease linker16 S rRNA
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