Table 4.
Analytical sensitivity and limit of detection; assay slope, linearity and efficiency were calculated for a quantitative range of 60–6 000 000 gene copies/reaction for all targets except for Pseudomonas aeruginosa, which had a quantitative range of 600–6 000 000 gene copies/reaction
| Number of replicates (%) detected for input bacterial spike, CFUs/reaction |
Limit of detection (95% level), CFUs/reaction | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Slope | Linearity (R2) | Efficiency | 100 000 CFUs/PCR (8 333 333 CFUs/mL) | 1000 CFUs/PCR (83 333 CFUs/mL) | 100 CFUs/PCR (8333 CFUs/mL) | 10 CFUs/PCR (833 CFUs/mL) | 1 CFU/PCR (83 CFUs/mL) | |||
| mRT-PCR 1 | Streptococcus pneumoniae | –3.18 | 0.92 | 1.06 | 12/12 (100) | 12/12 (100) | 12/12 (100) | 12/12 (100) | 2/9 (22) | 2.1 |
| Haemophilus influenzae | –3.11 | 0.97 | 1.10 | 12/12 (100) | 12/12 (100) | 12/12 (100) | 12/12 (100) | 12/12 (100) | 0.8 | |
| Staphylococcus aureus | –3.33 | 0.98 | 1.00 | 12/12 (100) | 12/12 (100) | 12/12 (100) | 7/9 (78) | 7/9 (78) | 15.5 | |
| Moraxella catarrhalis | –3.18 | 0.92 | 1.06 | 12/12 (100) | 12/12 (100) | 12/12 (100) | 6/12 (50) | 3/12 (25) | 23.1 | |
| mRT-PCR 2 | Escherichia coli | –3.27 | 0.95 | 1.05 | 12/12 (100) | 12/12 (100) | 11/11 (100) | 10/12 (83) | 8/12 (67) | 18.5 |
| Klebsiella pneumoniae | –3.43 | 0.93 | 1.00 | 12/12 (100) | 12/12 (100) | 11/11 (100) | 12/12 (100) | 8/12 (67) | 1.5 | |
| Pseudomonas aeruginosa | –3.46 | 0.96 | 0.96 | 12/12 (100) | 12/12 (100) | 10/11 (91) | 0/12 (0) | 0/12 (0) | 104.6 | |
| Acinetobacter baumannii | –3.20 | 0.96 | 1.08 | 12/12 (100) | 12/12 (100) | 11/11 (100) | 12/12 (100) | 5/12 (42) | 2.1 | |
| Control | Human | –3.37 | 0.99 | 0.98 | NA | NA | NA | NA | NA | NA |
mRT-PCR, multiplex real-time PCR; NA, not assessed.