This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X.
Right arrow Articles by Raskin, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X.
Right arrow Articles by Raskin, L.
Agricola
Right arrow Articles by Li, X.
Right arrow Articles by Raskin, L.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2008, p. 7297-7305, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01002-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons{triangledown} ,{dagger}

Xu Li,1 Eberhard Morgenroth,2,3 and Lutgarde Raskin1*

Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109,1 Department of Civil and Environmental Engineering,2 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 618013

Received 4 May 2008/ Accepted 16 September 2008

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 hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. 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.

* Corresponding author. Mailing address: University of Michigan, Department of Civil and Environmental Engineering, 107 EWRE Building, 1351 Beal Ave., Ann Arbor, MI 48109-2125. Phone: (734) 647-6920. Fax: (734) 763-2275. E-mail: raskin{at}umich.edu

{triangledown} Published ahead of print on 26 September 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, December 2008, p. 7297-7305, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01002-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»