Table 2.
Evaluation of Filtration methods to improve sensitivity and specificity of rt-qPCR assays
| Dilutions (3 × replicates) | Hydrolysis probe assay | SYBR Green assay | |||
|---|---|---|---|---|---|
| Crossing point (SD), Quant value (SD) | |||||
| Filtered | 5.0 × 105 | 23.38 (0.03), | 540 000 (9 000) | 20.95 (0.68) | 850 000 (400 000) |
| 1.0 × 105 | 25.36 (0.16) | 160 000 (16 000) | 24.20 (0.44) | 81 000 (26 000) | |
| 2.0 × 104 | 28.33 (0.17) | 27 000 (3000) | 26.14 (0.31) | 20 000 (4000) | |
| 1.0 × 103 | 33.74 (0.44) | 1000 (300) | 32.65 (0.49) | 200 (70) | |
| 5.0 × 102 | 34.64 (0.45) | 600 (160) | 33.29 (1.24) | 160 (130) | |
| 2.5 × 102 | 37.40 (n/a) | 110 (n/a) | 37.02 (1.05)a | 10 (10) | |
| 1.0 ×02 | Not detected | False Positive | 20 (10) | ||
| Unfiltered | 5.0 × 105 | 24.53 (0.62) | 300 000 (94 000) | 22.83 (0.46) | 200 000 (70 000) |
| 1.0 × 105 | 25.46 (0.35) | 156 000 (33 000) | 24.50 (0.11) | 64 000 (4800) | |
| 2.0 × 104 | 29.15 (0.05) | 17 000 (500) | 29.85 (0.44) | 1500 (500) | |
| 1.0 × 103 | 33.81 (0.38) | 1000 (220) | 32.98 (0.16) | 160 (20) | |
| 5.0 × 102 | 35.27 (1.63) | 550 (450) | 36.38 (0.23)a | 15 (3) | |
| 2.5 × 102 | 38.35 (1.06) | 75 (50) | 35.63 (0.73)a | 30 (10) | |
| 1.0 ×02 | Not detected | False Positive | 10 (2) | ||
a Indicates nonspecific melting peaks of 79°C obtained by melting curve analysis of replicates tested.
n/a = Not Applicable
SD = Standard Deviation
ND = Not Detected