Determination of detection limits, amplification efficiency of C. burnetii , correlation of automated and manual extraction. Probability of achieving a positive result (y-axis), depending on the DNA input copy number per mL EDTA blood (x-axis). A, Qiagen DNA mini kit; B, Qiagen M48 DNA mini kit, used on a Qiagen M48 automated DNA extraction instrument. Each datum point represents the rate of positive results in six replicate tests per concentration. Limits of detection are comparable with both methods of DNA extraction. C, Threshold cycles (y-axis) as a measure of efficiency of PCR amplification for C. burnetii and internal control. Each reaction contained 15 copies of plasmid-derived C. burnetii target gene and variable numbers of internal control plasmid pCoxmimic, as depicted on the x-axis. Results of eight replicate real-time PCR reactions per setting are shown as a result of box-plot analysis, showing the range of results by whiskers, whereby the two central quartiles of data are represented as a box. Solid line with grey boxes, C. burnetii target gene, broken line with white boxes, internal control. No reduced efficiency in amplification is observed for the C. burnetii target gene in presence of up to 100 copies of internal control. D, Correlation of C. burnetii DNA copies per ml as determined by C. burnetii real-time PCR after automated (x-axis) and manual extraction procedure (y-axis).