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Broad range DNA probes for detecting and amplifying eubacterial nucleic acids.

K Chen1, H Neimark, P Rumore

  • 1Department of Medicine, State University of New York, Health Science Center at Brooklyn 11203.

FEMS Microbiology Letters
|January 1, 1989
PubMed
Summary

New eubacterial 16S ribosomal RNA (rRNA) probes detect diverse bacteria in eukaryotic cells. These probes enable sensitive detection and partial phylogenetic classification of uncultured or unknown bacterial pathogens.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Eubacterial 16S ribosomal RNA (rRNA) sequences are conserved and essential for bacterial identification.
  • Detecting diverse eubacterial species, especially uncultured or unknown pathogens, remains a challenge in diagnostics.

Purpose of the Study:

  • To develop and characterize broadly homologous oligomer probes for detecting eubacterial 16S rRNA.
  • To assess the sensitivity and specificity of these probes in various molecular assays.

Main Methods:

  • Design and synthesis of two oligomer probes targeting conserved eubacterial 16S rRNA sequences.
  • Dot blot hybridization assays to test probe detection of diverse eubacterial nucleic acids.
  • Polymerase chain reaction (PCR) using probes as primers for enhanced sensitivity detection.

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Main Results:

  • Probes detected all 23 phylogenetically diverse eubacterial nucleic acids tested.
  • Sensitivity achieved was approximately 1 bacterium per 10 eukaryotic cells via dot blot.
  • PCR detected 1 pg of E. coli DNA in a large excess of eukaryotic DNA.
  • Direct sequence analysis of amplified DNA allowed partial phylogenetic classification.

Conclusions:

  • Broadly homologous probes offer advantages over specific probes for unknown organism detection.
  • These probes can identify bacterial infections in samples where organisms are uncultured or of unknown cause.
  • The developed probes show potential for broad-range bacterial detection and preliminary identification in complex samples.