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Molecular Biotechnology

, Volume 22, Issue 3, pp 231–242 | Cite as

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR

  • Sven KlaschikEmail author
  • Lutz E. Lehmann
  • Ansgar Raadts
  • Andreas Hoeft
  • Frank Stuber
Research

Abstract

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.

Index Entries

PCR bacteria 16S DNA decontamination UV-irradiation 8-methoxypsoralen DNAse restriction endonuclease digestion 

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Sven Klaschik
    • 1
    Email author
  • Lutz E. Lehmann
  • Ansgar Raadts
  • Andreas Hoeft
  • Frank Stuber
  1. 1.Klinik und Poliklinik für Anästhesiologie und Spezielle IntensivmedizinUniversität BonnBonnGermany

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