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Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria

A Vaid1, A H Bishop

  • 1School of Chemical and Life Sciences, University of Greenwich, London, UK.

Journal of Microbiological Methods
|October 16, 1999
PubMed

Insights

Optimizing DNA amplification in Gram-positive bacteria requires specific pre-treatments. Different bacterial species respond best to varied methods, with in situ PCR yielding the strongest fluorescence signals.

Area of Science:

  • Microbiology
  • Molecular Biology

Background:

  • Intracellular DNA amplification is crucial for microbial detection.
  • Optimizing permeabilization techniques is essential for accurate whole-cell analysis.

Purpose of the Study:

  • To optimize pre-treatment methods for intracellular DNA amplification in Gram-positive bacteria.
  • To compare the effectiveness of different amplification techniques: whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS.

Main Methods:

  • Bacteria were treated with paraformaldehyde, muramidases (mutanolysin, lysozyme), and mild acid hydrolysis.
  • Scanning electron microscopy and fluorescent photomicrography were used to assess treatment effectiveness and cell integrity.
  • In situ PCR, PRINS, and cycle PRINS were employed for DNA amplification and detection.

Main Results:

  • A combination of mutanolysin and lysozyme was optimal for Bacillus cereus.
  • Lysozyme alone was most effective for Streptomyces coelicolor, Lactococcus lactis, and Clostridium sporogenes.
  • Mycobacterium phlei showed the brightest fluorescence with direct amplification (no pre-treatment).
  • In situ PCR provided the strongest fluorescence signals compared to PRINS and cycle PRINS.

Conclusions:

  • Pre-treatment optimization is species-specific for effective intracellular DNA amplification.
  • In situ PCR is the most sensitive method among those tested for whole-cell bacterial analysis.

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