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Appl. Environ. Microbiol. doi:10.1128/AEM.01002-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI 48109; Department of Civil and Environmental Engineering, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^* To whom correspondence should be addressed. Email: raskin{at}umich.edu.

	Abstract

The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridization to non-target 16S rRNA. A concentration of 10 mM NaCl in the hybridization buffer was found optimal in maximizing the difference between final hybridization signals from target and non-target 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to non-target 16S rRNA. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.