Table 1.
Analysis of assay performance using dT1 templates and AmpliTaq Gold™ polymerase or VWR® TEMPase Hot Start DNA Polymerase
| Recovery | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Assay conditions | qPCR | Data acquisition | T (°C) | Calibration range (pmol) | R 2 | LODa (pmol) | LOQa (pmol) | Accuracy low, high (%) | Interassay CV (%) | Intraassay CV (%) | (%) | CFU or cellb / | Sample type | |
| dTTP | 20 pmol TPP, 0.875 u AmpliTaq Gold™, 5 mM MgCl2 | BioRad CFX96 | 14 s for 600 cycles | 60 | 0.63–12 | 0.986 ± 0.006 | 0.41 ± 0.07 | 0.68 ± 0.12 | 100 ± 14 100 ± 6 |
9.6 ± 7.0 | 7.8 ± 5.3 | 105 | 1 × 107 | M. smegmatis |
| 100 | 1 × 108 | S. aureus | ||||||||||||
| 98 | 2 × 105 | MES-SA | ||||||||||||
| 10 pmol TPP 0.9 unit VWR® TEMPase, 2 mM MgCl2 | QuantStudio 1 | 13 s for 260 cycles | 55 | 0.1–6 | 0.999 | 0.05 | 0.10 | 102 ± 7 109 ± 4 |
5.5 ± 6.5 | 5.3 ± 3.7 | 101 | 1 × 105 | MES-SA | |
| dCTP | 10 pmol TPP, 0.438 u AmpliTaq Gold™, 2.5 mM MgCl2 | BioRad CFX96 | 13 s for 60 s then 80 s for 128 cycles | 60 | 1–8 | 0.991 ± 0.001 | 0.70 ± 0.13 | 1.10 ± 0.03 | 98 ± 4 100 ± 5 |
7.4 ± 4.3 | 6.9 ± 4.6 | 98 | 5 × 106 | M. smegmatis |
| 76 | 1 × 107 | M. smegmatis | ||||||||||||
| 92 | 1 × 108 | S. aureus | ||||||||||||
| 94 | 1 × 106 | MES-SA | ||||||||||||
| 10 pmol TPP, 0.9 u VWR® TEMPase, 2 mM MgCl2 | QuantStudio 1 | 13 s for 260 cycles | 55 | 0.6–10 | 0.992 | 0.30 | 0.50 | 101 ± 3 100 ± 9 |
6.5 ± 4.4 | 5.2 ± 2.9 | 92 | 6 × 107 | M. smegmatis | |
| dGTP | 10 pmol TPP, 0.438 u AmpliTaq Gold™, 2.5 mM MgCl2 | BioRad CFX96 | 13 s for 60, then 80 s for 128 cycles | 60 | 0.5–10 | 0.992 ± 0.001 | 0.50 ± 0.07 | 0.83 ± 0.11 | 99 ± 21 99 ± 4 |
12.6 ± 10.2 | 9.4 ± 5.7 | 102 | 1 × 107 | M. smegmatis |
| 109 | 5 × 105 | MES-SA | ||||||||||||
| 10 pmol TPP, 0.438 u AmpliTaq Gold™, 5 mM MgCl2 | QuantStudio 1 | 13 s for 100, then 80 s, for 200cycles | 57 | 0.25–8 | 0.997 | 0.18 | 0.33 | 99 ± 15 98 ± 6 |
ND | ND | 115 | 5 × 107 | E. coli | |
| 91 | 4 × 107 | S. aureus | ||||||||||||
| 10 pmol TPP, 0.9 u VWR® TEMPase, 3.5 mM MgCl2 | QuantStudio 1 | 13 s for 260 cycles | 55 | 0.4–10 | 0.999 | 0.11 | 0.19 | 99 ± 2 101 ± 5 |
5.6 ± 4.9 | 4.5 ± 2.5 | 101 | 6 × 106 | M. smegmatis | |
| dATP | 20 pmol TPP, 0.875 u AmpliTaq Gold™, 5 mM MgCl2 | BioRad CFX96 | 17 s for 700 cycles | 60 | 1–8 | 0.991 ± 0.004 | 0.59 ± 0.26 | 1.06 ± 0.26 | 95 ± 7 100 ± 0.1 |
12.0 ± 0.1 | 7.9 ± 1.6 | 92 | 1 × 106 | MES-SA |
| 73 | 1 × 108 | S. aureus | ||||||||||||
| 102 | 1 × 107 | M. smegmatis | ||||||||||||
| 15 pmol TPP, 0.9 u VWR® TEMPase, 5 mM MgCl2 | BioRad CFX96 | 13 s for 260 cycles | 60 | 1.5–15 | 0.977 | 1.68 | 2.81 | 104 ± 3 99 ± 1 |
10.1 ± 2.4 | 3.3 ± 1.8 | 97 | 1 × 108 | M. smegmatis | |
aLOD and LOQ were calculated by the two methods presented in the methods sections. If the mathematical calculation of LOD and LOQ gave lower value than the lowest resolvable calibration point, then the lowest calibration point should be considered as the LOD.
bdNTP extract from the given number of CFU (for bacteria) or cells (for eukaryotic cells) / well.
TPP: template, probe, primer (NDP1), u: unit.