Table 2.
Intra- and inter-assay reproducibility of HPV16 (a) and HPV18 (b) qPCR multiplexes for selected primer and probe concentrations.
| Probe concentrations | Primer concentrations | E6 | E7 | GAPDH | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Coefficient of variation | Coefficient of variation | Coefficient of variation | |||||||||||
| Ct meana | RFUs mean | Intra-assay | Inter-assay (%) | Ct meana | RFUs mean | Intra-assay | Inter-assay (%) | Ct meana | RFUs mean | Intra-assay | Inter-assay (%) | ||
| (a) HPV16 | |||||||||||||
| 50 nM | 400 nM | 24.13 | 809 | 0.05% ≤ CV ≤ 1.7% | 0.24 | 23.31 | 417 | 0.94% ≤ CV ≤ 1.81% | 0.84 | 22.05 | 265 | 0.47% ≤ CV ≤ 1.82% | 1.30 |
| 600 nM | 24.35 | 826 | 0.35% ≤ CV ≤ 0.92% | 0.91 | 23.46 | 427 | 0.88% ≤ CV ≤ 1.70% | 0.54 | 22.41 | 257 | 0.00% ≤ CV ≤ 0.81% | 0.32 | |
| 800 nM | 24.44 | 823 | 0.83% ≤ CV ≤ 0.97% | 0.70 | 23.42 | 433 | 1.34% ≤ CV ≤ 3.34% | 1.39 | 22.47 | 260 | 0.55% ≤ CV ≤ 1.36% | 1.15 | |
| 1000 nM | 24.41 | 823 | 0.24% ≤ CV ≤ 0.42% | 0.81 | 23.52 | 430 | 0.39% ≤ CV ≤ 3.17% | 0.63 | 22.48 | 254 | 0.27% ≤ CV ≤ 0.52% | 0.08 | |
| 100 nM | 400 nM | 24.11 | 1892 | 0.52% ≤ CV ≤ 3.30% | 0.80 | 23.35 | 951 | 0.35% ≤ CV ≤ 7.38% | 1.90 | 22.64 | 616 | 1.45% ≤ CV ≤ 2.89% | 0.83 |
| 600 nM | 24.27 | 1770 | 0.18% ≤ CV ≤ 0.41% | 0.45 | 23.06 | 945 | 0.98% ≤ CV ≤ 2.21% | 0.15 | 22.54 | 582 | 0.40% ≤ CV ≤ 0.58% | 0.81 | |
| 800 nM | 24.35 | 1726 | 0.21% ≤ CV ≤ 0.78% | 0.75 | 23.00 | 937 | 0.91% ≤ CV ≤ 1.29% | 1.84 | 22.44 | 564 | 0.59% ≤ CV ≤ 2.37% | 1.63 | |
| 1000 nM | 24.33 | 1731 | 0.24% ≤ CV ≤ 0.84% | 0.55 | 23.02 | 927 | 1.11% ≤ CV ≤ 1.54% | 0.06 | 22.51 | 555 | 0.34% ≤ CV ≤ 1.53% | 1.04 | |
| 150 nM | 400 nM | 24.19 | 2435 | 0.10% ≤ CV ≤ 0.42% | 0.11 | 23.23 | 1311 | 1.58% ≤ CV ≤ 1.79% | 1.63 | 22.50 | 827 | 0.54% ≤ CV ≤ 0.90% | 1.20 |
| 600 nM | 24.19 | 2465 | 0.25% ≤ CV ≤ 0.66% | 0.26 | 23.23 | 1316 | 0.26% ≤ CV ≤ 2.98% | 1.41 | 22.58 | 800 | 0.52% ≤ CV ≤ 0.81% | 1.18 | |
| 800 nM | 24.37 | 2496 | 0.47% ≤ CV ≤ 1.30% | 0.17 | 23.35 | 1340 | 0.86% ≤ CV ≤ 1.60% | 1.58 | 22.43 | 807 | 0.41% ≤ CV ≤ 1.62% | 2.90 | |
| 1000 nM | 24.28 | 2508 | 0.17% ≤ CV ≤ 0.31% | 0.90 | 23.39 | 1341 | 0.17% ≤ CV ≤ 1.79% | 1.99 | 22.63 | 799 | 0.15% ≤ CV ≤ 0.59% | 1.51 | |
| 200 nM | 400 nM | 24.29 | 3279 | 0.12% ≤ CV ≤ 0.94% | 0.76 | 23.30 | 1747 | 0.25% ≤ CV ≤ 2.07% | 0.63 | 22.93 | 1143 | 0.78% ≤ CV ≤ 1.14% | 1.19 |
| 600 nM | 24.35 | 3235 | 0.04% ≤ CV ≤ 0.62% | 0.43 | 23.45 | 1694 | 0.42% ≤ CV ≤ 4.02% | 0.90 | 22.79 | 1079 | 0.48% ≤ CV ≤ 1.67% | 0.86 | |
| 800 nM | 24.33 | 3309 | 0.17% ≤ CV ≤ 1.86% | 0.89 | 22.89 | 1832 | 0.37% ≤ CV ≤ 3.18% | 3.28 | 22.52 | 1130 | 0.22% ≤ CV ≤ 1.05% | 0.40 | |
| 1000 nM | 24.40 | 3156 | 0.25% ≤ CV ≤ 0.54% | 0.30 | 23.04 | 1728 | 0.55% ≤ CV ≤ 2.43% | 0.90 | 22.66 | 1052 | 0.17% ≤ CV ≤ 2.22% | 2.01 | |
| (b) HPV18 | |||||||||||||
| 50 nM | 400 nM | 22.70 | 790 | 0.27% ≤ CV ≤ 1.14% | 1.47 | 24.81 | 497 | 0.62% ≤ CV ≤ 2.91% | 0.71 | 22.44 | 273 | 0.58% ≤ CV ≤ 2.11% | 0.72 |
| 600 nM | 22.85 | 848 | 0.65% ≤ CV ≤ 1.13% | 1.13 | 24.77 | 510 | 0.20% ≤ CV ≤ 2.29% | 0.91 | 22.60 | 256 | 0.20% ≤ CV ≤ 1.89% | 1.01 | |
| 800 nM | 23.10 | 908 | 0.09% ≤ CV ≤ 0.47% | 0.28 | 24.79 | 542 | 0.68% ≤ CV ≤ 2.14% | 1.86 | 22.68 | 268 | 0.26% ≤ CV ≤ 1.28% | 1.77 | |
| 1000 nM | 23.26 | 835 | 0.24% ≤ CV ≤ 1.04% | 1.44 | 24.75 | 507 | 0.60% ≤ CV ≤ 1.86% | 1.36 | 22.78 | 237 | 0.26% ≤ CV ≤ 1.06% | 1.36 | |
| 100 nM | 400 nM | 22.33 | 1768 | 0.56% ≤ CV ≤ 1.74% | 3.17 | 24.83 | 1025 | 0.44% ≤ CV ≤ 5.48% | 3.13 | 22.93 | 610 | 0.09% ≤ CV ≤ 1.54% | 2.22 |
| 600 nM | 22.79 | 1791 | 0.30% ≤ CV ≤ 0.62% | 2.94 | 24.84 | 995 | 0.85% ≤ CV ≤ 3.24% | 1.16 | 22.98 | 581 | 0.44% ≤ CV ≤ 1.24% | 2.50 | |
| 800 nM | 22.74 | 1877 | 0.60% ≤ CV ≤ 1.70% | 3.73 | 24.69 | 1016 | 0.53% ≤ CV ≤ 0.69% | 1.38 | 22.90 | 582 | 0.31% ≤ CV ≤ 1.70% | 3.32 | |
| 1000 nM | 22.97 | 1846 | 0.48% ≤ CV ≤ 1.54% | 3.48 | 25.00 | 968 | 0.86% ≤ CV ≤ 2.23% | 2.09 | 23.06 | 552 | 0.28% ≤ CV ≤ 1.51% | 2.96 | |
| 150 nM | 400 nM | 22.91 | 2115 | 0.39% ≤ CV ≤ 1.30% | 1.06 | 24.81 | 1270 | 0.43% ≤ CV ≤ 2.61% | 1.65 | 23.25 | 747 | 0.15% ≤ CV ≤ 2.11% | 0.71 |
| 600 nM | 23.18 | 2313 | 0.26% ≤ CV ≤ 0.66% | 1.07 | 25.50 | 1265 | 1.19% ≤ CV ≤ 7.30% | 1.82 | 23.32 | 730 | 0.45% ≤ CV ≤ 1.00% | 0.89 | |
| 800 nM | 23.21 | 2366 | 0.29% ≤ CV ≤ 1.82% | 0.56 | 24.90 | 1347 | 1.13% ≤ CV ≤ 3.23% | 0.89 | 23.15 | 703 | 0.30% ≤ CV ≤ 1.37% | 0.33 | |
| 1000 nM | 23.36 | 2485 | 0.20% ≤ CV ≤ 1.35% | 0.99 | 25.10 | 1384 | 0.62% ≤ CV ≤ 4.09% | 0.43 | 23.35 | 725 | 0.85% ≤ CV ≤ 1.56% | 0.70 | |
| 200 nM | 400 nM | 23.01 | 2948 | 0.24% ≤ CV ≤ 1.09% | 0.98 | 25.15 | 1687 | 1.34% ≤ CV ≤ 1.92% | 0.83 | 23.36 | 1107 | 0.28% ≤ CV ≤ 0.64% | 0.96 |
| 600 nM | 23.19 | 3298 | 0.33% ≤ CV ≤ 0.61% | 0.64 | 25.45 | 1629 | 0.46% ≤ CV ≤ 2.32% | 1.20 | 25.52 | 1075 | 0.20% ≤ CV ≤ 0.94% | 1.15 | |
| 800 nM | 23.33 | 3499 | 0.28% ≤ CV ≤ 0.60% | 0.76 | 24.96 | 1734 | 0.36% ≤ CV ≤ 1.01% | 1.78 | 23.49 | 1084 | 0.64% ≤ CV ≤ 0.88% | 0.15 | |
| 1000 nM | 23.51 | 3354 | 0.21% ≤ CV ≤ 1.16% | 1.06 | 25.05 | 1702 | 0.98% ≤ CV ≤ 1.18% | 1.69 | 23.65 | 1,000 | 0.14% ≤ CV ≤ 0.87% | 0.58 | |
Ct cycle threshold.
aMean of inter-assay Ct from technical triplicate.