Molecular Biotechnology

, Volume 7, Issue 3, pp 207–216 | Cite as

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

  • Farida Hilali
  • Patrick Saulnier
  • Elisabeth Chachaty
  • Antoine AndremontEmail author


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

PCR polymerase DNase 16S rRNA sequences bacteria 


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Copyright information

© Humana Press Inc 1997

Authors and Affiliations

  • Farida Hilali
    • 1
  • Patrick Saulnier
    • 1
  • Elisabeth Chachaty
    • 1
  • Antoine Andremont
    • 2
    Email author
  1. 1.Laboratoire de Microbiologie MédicaleInstitut Gustave-RoussyVillejuifFrance
  2. 2.Laboratoire de BactériologieGroupe Hospitalier Bichat-Claude BernardParis CEDEX 18France

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