Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

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.

Performance evaluation of the Verigene® (Nanosphere) and FilmArray® (BioFire®) molecular assays for identification of causative organisms in bacterial bloodstream infections

“…False-positive tests resulting in pseudo-outbreaks due to contamination of blood culture bottles have previously been described [24,25], as has contamination of molecular reagents [26]. Evaluation by other authors of the FilmArray® Blood Culture Identification Panel have not identified this issue [27].…”

Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using Sonication

“…Taq DNA polymerases and other PCR reagents may be contaminated with bacterial DNA (22). Strategies to eliminate spurious bacterial DNA include treatment of reagents with gamma irradiation (6,9), UV irradiation (6), restriction endonucleases (6), DNase I (6), or heat-labile double-strand-specific DNase (Biotec Marine Biochemicals, Tromsø, Norway) (6); reported results have been variable.…”

Accuracy, Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi

“…When E. coli or universal bacterial primers are applied to no-template controls, qPCR typically produces a positive signal ranging from 10 1 to 10 2 gene copies. In these cases, custom qPCR master mixes may be produced in order to independently dilute the DNA polymerase concentration (51), which, in turn, reduces the signal in no-template controls. This reduction, however, changes the shape of qPCR curves plotting fluorescence versus cycle number and requires that full standard curves be produced at these reduced DNA polymerase amounts.…”