Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons

Xu Li, Eberhard Morgenroth, Lutgarde Raskin

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)7297-7305
Number of pages9
JournalApplied and environmental microbiology
Volume74
Issue number23
DOIs
StatePublished - Dec 1 2008

Fingerprint

Peptide Nucleic Acids
nucleic acid
peptide
nucleic acids
hybridization
ribosomal RNA
assay
peptides
assays
Dechlorosoma suillum
Molecular Probes
RNA
Buffers
Dechlorosoma
probes (equipment)
Dechloromonas
Bioreactors
buffers
Protein Sorting Signals
Complex Mixtures

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons. / Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde.

In: Applied and environmental microbiology, Vol. 74, No. 23, 01.12.2008, p. 7297-7305.

Research output: Contribution to journalArticle

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