001 ++− 0.008 +−− 0.077 — 0.744 5 μl Reaction   +++ 0.006 ++− 0.026 +−− 0.120 — 0.557 FungiQuant amplification and RXDX-101 clinical trial quantitative profiles against pure plasmids, C. albicans DNA, and templates with background human DNA We showed FungiQuant had

excellent amplification profiles against C. albicans plasmid standards and C. albicans DNA, with quantitative dynamic RG7420 molecular weight range of 25 – 107 copies and 10 fg – 10 ng C. albicans DNA, respectively (Figure 2A-B). A list of fungal species that are perfect matches to C. albicans in the FungiQuant primer and probe region can be found in Additional file 5: Table S6. Figure 2 A-B. FungiQuant amplification profiles. The FungiQuant amplification profiles remain consistent, irrespective of reaction volume and type of DNA template. The amplification profiles of

plasmid standards (Fig. 2 A) and C. albicans DNA (Fig. 2 B) in two reaction volumes (5 μl and 10 μl) are presented. We also showed that FungiQuant had strong www.selleckchem.com/products/a-1210477.html reproducibility, even when we added background human DNA. The inter-run coefficients of variance (CoV) ranged from 0.37 – 3.80% and 3.52 – 34.39% for Ct-value and copy number, respectively. The intra-run average CoV were 0.35 – 2.90% and 1.98 – 23.74% Ct-value and copy number, respectively (Figure 3, Additional file 6: Figure S2). We found that 5 μl reactions had greater inter-run CoV than 10 μl reactions (Figure 3). This suggests that the 10 μl reaction volumes is better suited for quantitative use. Figure 3 A-B. FungiQuant inter- and intra-run coefficient of variation (CoV). FungiQuant CoV is presented for copy number (solid line) and Ct-value Florfenicol (dashed line), demonstrating the range of CoV, which is lower for the 10 μl than the 5 μl reactions. For the 10 μl reactions, the FungiQuant intra-run copy number CoV is consistently below 15% until at 25 copies, and for the 5 μl reactions,

the intra-run CoV is below 20% until at 50 copies. The FungiQuant Ct-value CoV is consistently below 10%, irrespective of reaction volumes. We further determined that FungiQuant’s amplification profile and assay dynamic range were not impacted by the presence of human DNA, at up to 10 ng (Table 4, Additional file 7: Figure S3A-D). Thus, FungiQuant is robust quantitatively even when the fungal 18S rRNA gene is relatively rare as compared to background human DNA. Specifically, we showed that FungiQuant could be applied quantitatively at a ratio of 25:679,464 fungal-to-human 18S rRNA gene copy number. FungiQuant is robust for low number of fungal 18S rRNA gene To validate FungiQuant use for samples with low fungal DNA and high human DNA, we developed guidelines for interpreting triplicate reactions. Additional file 1: Table S2 provides the sensitivity and specificity results from FungiQuant evaluation against multiple positive and negative controls in 10 μl and 5μl reaction volumes. Our analysis showed that FungiQuant could consistently detect 5 copies of 18S rRNA gene template, whereas 1.