Abstract
BackgroundPCR 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 FindingsThis 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 (H2O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase.Conclusions/SignificanceIt 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.
Highlights
PCR in principle can detect a single target molecule in a reaction mixture
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
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
Summary
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. Trace bacterial DNA in Taq polymerase is hard to eliminate because of the enzyme’s bacterial source as well as reagents and equipment used in its purification. This is a problem for the detection of bacterial DNA when two specific conditions apply: 1) Target DNA is present in very small quantities; and 2) Bacterial species are unknown, possibly mixed, and the assay cannot be designed to exclude specific species. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. All commercial Taq polymerase preparations inevitably contain contaminating microbial DNA.
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