Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria

PLoS One. 2009 Sep 15;4(9):e7010. doi: 10.1371/journal.pone.0007010.

Abstract

Background: PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical.

Methodology/principal findings: This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H(2)O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase.

Conclusions/significance: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved "treatment-free" attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Typing Techniques / methods
  • Base Sequence
  • Cloning, Molecular
  • DNA / analysis
  • DNA / isolation & purification
  • DNA / metabolism
  • DNA Restriction Enzymes / metabolism
  • DNA, Bacterial / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Pseudomonas fluorescens / genetics
  • RNA, Ribosomal, 16S / chemistry
  • Sequence Homology, Nucleic Acid
  • Taq Polymerase / chemistry*
  • Taq Polymerase / metabolism
  • beta-Lactamases / metabolism

Substances

  • DNA, Bacterial
  • RNA, Ribosomal, 16S
  • DNA
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • DNA Restriction Enzymes
  • beta-Lactamases