Molecular Biotechnology

, Volume 7, Issue 3, pp 207–216

Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes

Authors

  • Farida Hilali
    • Laboratoire de Microbiologie MédicaleInstitut Gustave-Roussy
  • Patrick Saulnier
    • Laboratoire de Microbiologie MédicaleInstitut Gustave-Roussy
  • Elisabeth Chachaty
    • Laboratoire de Microbiologie MédicaleInstitut Gustave-Roussy
    • Laboratoire de BactériologieGroupe Hospitalier Bichat-Claude Bernard
Research

DOI: 10.1007/BF02740812

Cite this article as:
Hilali, F., Saulnier, P., Chachaty, E. et al. Mol Biotechnol (1997) 7: 207. doi:10.1007/BF02740812

Abstract

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNase I and was followed by DNase inactivation at 94°C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase, Deep-Vent Exo-®polymerase (New England Biolabs, Beverly, MA), and super-Taq® polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq® polymerase (Perkin-Elmer Cetus, Norwalk, CT), Ampli-Taq LD® polymerase (Perkin-Elmer Cetus) or Deep-vent® polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.

Index Entries

PCRpolymeraseDNase16S rRNA sequencesbacteria
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Copyright information

© Humana Press Inc 1997