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Identification of indicator microorganisms using a standardized PNA FISH method.

H Perry-O'Keefe1, S Rigby, K Oliveira

  • 1Boston Probes, Bedford, MA, USA. hokeefe@bostonprobes.com

Journal of Microbiological Methods
|November 21, 2001
PubMed
Summary

A new Peptide Nucleic Acid (PNA) FISH method enables simultaneous identification of multiple bacteria and yeast species. This advancement offers improved diagnostics for clinical and industrial microbiology applications.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Accurate identification of microorganisms is crucial for clinical diagnostics and industrial processes.
  • Traditional methods for microbial identification can be time-consuming and lack specificity.
  • Fluorescent in situ hybridization (FISH) offers a promising alternative for rapid microbial detection.

Purpose of the Study:

  • To develop and standardize a Peptide Nucleic Acid (PNA) FISH method for analyzing gram-negative and gram-positive bacteria, and yeast.
  • To design and evaluate PNA probes targeting specific rRNA sequences of key bacterial and eukaryotic species.
  • To demonstrate the potential of multiplex PNA FISH for simultaneous identification of multiple microbial species in mixed populations.

Main Methods:

Related Experiment Videos

  • Development of fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella, eubacteria, and eucarya.
  • Evaluation of PNA probes using PNA FISH on 27 bacterial species and 1 yeast species.
  • Assessment of probe specificity and cross-reactivity.
  • Demonstration of multiplex FISH using four-color imaging for simultaneous identification of multiple species.

    • Main Results:

    • PNA probes for S. aureus and P. aeruginosa showed high specificity, with no cross-reactivity observed.
    • The E. coli PNA probe detected Shigella species, as predicted by sequence data.
    • The Salmonella PNA probe identified all tested Salmonella subspecies but also reacted with a few other bacterial species.
    • Eubacteria- and eucarya-specific PNA probes successfully detected all bacterial and yeast species, respectively.
    • Simultaneous identification of E. coli, P. aeruginosa, S. aureus, and Salmonella in mixed populations was achieved using multiplex FISH.

    Conclusions:

    • The developed PNA FISH method provides a standardized and reliable approach for microbial identification.
    • PNA FISH offers superior specificity and the capability for simultaneous detection of multiple microbial species compared to DNA probes.
    • This multiplex PNA FISH technique holds significant potential for diverse diagnostic applications in clinical and industrial microbiology.