3, the triplicates were compared and if a clear outlier was present (ΔCt > 0.3 from other two replicates), the outlier well was excluded from analysis. Amplification profiles of the seven conditions tested were annotated and presented in Figure2A-B and Additional file 4: Figure S 4A-E. Results from laboratory quantitative validation using all conditions tested were summarized PD0332991 concentration in Table4. Detailed results of inter- and intra-run

coefficient of variation for Ct value and copy number were presented for all conditions tested in Figure3 and Additional file 5: Supplemental file 1A-C using scattered plots generated with the vegan package in R [18, 19]. Figure 2 A-B. Standard curve amplification profiles of the BactQuant assay generated from 10 μl and 5 μl reactions using seven ten-fold dilutions and normalized plasmid standards at 10 9 copies/μl. The Ct value of standard curve using 5 μl reaction volumes (Figure2B) shows an approximately 1 Ct left shift from the 10 μl reaction volumes LDC000067 clinical trial (Figure2A). However, the overall amplification profiles are not significantly different between the different reaction volumes over the assay dynamic range of 102 Selleck CBL0137 copies to 108 copies of 16 S rRNA gene per reaction. Table 4 Laboratory quantitative validation results of the BactQuant assay performed using pure plasmid standards and different mixed templates Templates used Assay dynamic range Average

reaction efficiency (SD) r 2 –value Plasmid standards–only (10 μl Rxn) 100–108 copies 102% (2%) >0.999 Plasmid standards-only (5 μl Rxn) 100 – 108 copies 95% (1%) >0.999 Plasmid standards plus 0.5 ng human gDNA 100 – 108 copies 99% (4%) >0.994 Plasmid standards plus 1 ng human gDNA 100 – 108 copies 101% (5%) >0.994 Sulfite dehydrogenase Plasmid standards plus 5 ng human gDNA 500 – 108 copies 96% (1%) >0.999 Plasmid standards plus 10 ng human gDNA 1000 – 108 copies 97% (2%) >0.999 Plasmid standards plus 0.5 ng  C. albicans gDNA 100 – 108 copies 97% (1%) >0.999

Figure 3 Inter- and intra-run coefficient of variation (CoV) for 10 μl and 5 μl reactions using seven ten-fold dilutions and normalized plasmid standards at 10 9 copies/μl calculated using data from multiple runs. The data is presented for both copy number ( solid line) and Ct value ( dashed line). As would be expected, the CoV is higher for copy number than for Ct value and is also higher for inter-run than for intra-run. The CoV for copy number for both reaction volumes was consistently below 15% until at 107 copies for 5 μl reactions. The CoV for Ct value was consistently below 5% for both reaction volumes. Bacteria-to-human ratio calculations Calculations were performed using the following copy number and genome size estimates: the average bacterial 16 S rRNA gene copy number per genome was estimated to be 3.94 copies as calculated by rrnDB [20] (accessed at http://​ribosome.​mmg.​msu.​edu/​rrndb/​index.​php) and the average human 18 S rRNA gene copy number per genome was estimated to be 400 copies [21].