Figure 1.
Flowchart of experimental set-up.
SA, S. aureus; PA, P. aeruginosa; CA, C. albicans; CFU, colony forming unit.
Figure 2.
Overview of the Polaris method.
Whole blood is depicted, consisting of human cells and DNA, and some pathogens. In the first step, human cells and DNA are degraded and pathogens remain intact. In the second step, intact pathogens are pelleted by centrifugation. Finally, this pellet is washed and pathogens are lysed. Subsequently, DNA can be isolated (not depicted). WBC, white blood cell; RBC, red blood cell; SLB, selective lysis buffer; BLB, bug lysis buffer.
Table 1.
Overview of primers and probes used for pathogen detection.
Figure 3.
Comparison of Polaris and MolYsis methods using 1 ml and 5 ml spiked whole blood samples.
The grey bars represent the Polaris samples (1 or 5 ml whole blood), and the white bars represent the MolYsis isolated samples (1 or 5 ml whole blood). SEM is shown. The numbers in the bars represent the sample numbers.
Figure 4.
Polaris pathogen DNA isolation from reference (PBS) compared to 5 ml whole blood.
The indicated pathogens were spiked in 5 ml whole blood or processed as reference samples as described in the Materials and Methods. For all pathogens similar Ct values were obtained when isolated from PBS or whole blood. The grey bars represent the spiked 5 ml whole blood samples and the white bars the reference samples. SEM is shown. The numbers in the bars represent the sample numbers.
Figure 5.
Human DNA removal by different procedures.
Ct value comparison of the RNAseP PCR for all pathogen DNA isolation procedures. RNAseP is a marker to measure human DNA removal after pathogen DNA isolation. Standard deviations are shown of at least 6 independent experiments. One-way ANOVA analysis indicated that TTE-EasyMAG removes the least amount of human DNA as compared to all other methods (p<0.001), except when compared to MolYsis 1 ml (p = 0.156). MolYsis 5 ml removes most human DNA as compared to all other methods (p<0.000). Significant differences were also found when comparing Polaris 1 ml with both MolYsis 1 ml (p = 0.002) and 5 ml (p<0.000), and when comparing MolYsis 1 ml with MolYsis 5 ml (p<0.000).
Table 2.
Detection rates (percentage of positive PCRs) of 3 different DNA isolation methods in dilutions series of 1–1000 CFU/ml.