Improved Templated Fluorogenic Probes Enhance the Analysis of Closely Related Pathogenic Bacteria by Microscopy and Flow Cytometry

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• 作者：Raphael M. Franzini ; Eric T. Kool
• 刊名：Bioconjugate Chemistry
• 出版年：2011
• 出版时间：September 21, 2011
• 年：2011
• 卷：22
• 期：9
• 页码：1869-1877
• 全文大小：947K
• 年卷期：v.22,no.9(September 21, 2011)
• ISSN：1520-4812

文摘

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.