Different concentrations of purified, genomic DNA from F. tularensis SCHU S4 were spiked into dilutions of the seven different matrices with LODs determined using real-time PCR to identify the lowest amount of Indibulin template detectable at the highest concentration of sample matrix. Not surprisingly, different chemistries worked better for different types of inhibitory matrices. Surprisingly, we did observe a fluorescence signal when these enzymes were used without Taq. While this was unexpected since there is no 5���C3�� exonuclease activity, we hypothesized that the polymerase was priming off of the probe annealed to the template, and the strong 3���C5�� exonuclease activity was hydrolyzing the 3�� end of the probe to generate the observed signal. Data generated using gel-purified amplicon and excess probe without any primers suggest this hypothesis is correct as fluorescence was observed in template concentration-dependent fashion. Of note, these and almost all subsequent experiments with inhibitor-resistant PCR reagents showed less than ideal realtime PCR kinetics with amplification of the target, i.e. nonsigmoidal amplification, wavering baseline, and inconsistent endpoint fluorescence. Because the qualitative aspect and visualization of the realtime PCR kinetics increases confidence in software-based adjudication of Cq values, we scored reactions using a qualitative 1 to 5 scale with 5 being ideal and 1 being unacceptable. The principal reason IDE 1 nucleic acids are purified from clinical and environmental samples prior to PCR is to remove a significant amount of PCR inhibitors in these samples. This purification process can be highly inefficient as we previously observed with DNA purification using chaotropic lysis and silica-based nucleic acid binding. While purification would be appropriate for signatures at sufficient concentrations, this approach might be suboptimal for rare nucleic acid signatures, especially for pathogen detection. Also, specific extraction processes are typically applied based on sample matrix and/ target organism. This study sought to investigate direct PCR assessment in clinical and environmental samples using inhibitor-resistant PCR reagents as a mechanism to increase the sensitivity of real-time PCR assays as well as remove sample-specific sample preparation. Given the manufacturer-described functionality, reagents designed for PCR amplification directly in clinical and environmental samples were selected and evaluated. Generally, the polymerases tested did not generate a sufficient fluorescence signal when used alone, likely due to the absence of 5���C3�� exonuclease activity in all of the polymerases except for the KAPA polymerase. This 5���C3�� exonuclease function is a critical component for probe-based hydrolysis. Inclusion of Taq polymerase, having this exonuclease activity, improved probe hydrolysis detection. Interestingly, all but one of the inhibitor-resistant enzymes produced some baseline targetspecific detection without the addition of native Taq polymerase.