This may be overcome by using an intercalating reporter dye in place of a fluorescent probe as a qPCR reporter

mechanism; however, the loss of tertiary-level of specificity is a potential concern in direct application of an intercalating dye assay to specimens containing high amounts of nontarget DNA. Exogenous bacterial DNA, particularly from biologically synthesized reagents such as Taq DNA polymerase are a known limitation for analyzing samples with low bacterial load [28, 33]. Recently, this issue has received renewed attention due to increased usage of next-generation sequencing and the frequent data contamination from exogenous bacterial DNA. Several methods have been evaluated for removing bacterial contaminants from Taq DNA polymerase, including UV irradiation [34, 35], DNAse I treatment, and ultrafiltration [36]. The level of E. coli contamination in Taq DNA polymerase has been estimated at 102 to 105 genome Trametinib equivalents MAPK Inhibitor Library ic50 of bacterial DNA per unit of enzyme [28]. This is consistent with the lowest amount of contamination we have observed in our experiments, which were 5 and 10 copies of 16 S rRNA gene in 5 μl and 10 μl reactions, respectively. The ubiquity of bacterial DNA also makes the determination of assay specificity challenging. Our use of qPCR-quantified plasmid standards addressed a major limitation in the preparation of qPCR quantification standards. The

conventional approach of quantifying bacterial genomic DNA or plasmid Avelestat (AZD9668) standards necessitates converting mass (i.e., nanograms per μl) to copy number (i.e., 108 copies per μl) and can introduce substantial error. Thus far, we have also successfully applied BactQuant to a diverse range of clinical specimens, including swab eluents, surgical specimens, and respiratory specimens, but we did not present these findings in this paper. To fully understand the likelihood of false negative results

due to interference from human DNA or BactQuant’s limit of detection will require additional evaluations. Conclusion In summary, we have developed and evaluated a new broad-coverage qPCR assay—BactQuant—for bacterial detection and quantification that showed concurrently improved assay coverage and favorable quantitative parameters. Laboratory tests showed that in vitro performance was even better than predicted in the in silico analysis; however, our approach of evaluating assay coverage by considering the primer and probe sequences as a single unit is appropriate and necessary. In addition, when employing a copy number estimation method, such as qPCR, the quantification of standards is critical for accurate template quantification. Thus, our approach of quantifying plasmid standards using the intrinsic property of real-time PCR is another important step for any absolute quantification experiments using qPCR.