Table 2.
Low target detection and mismatch tolerance in real-time RT-PCR for different probe designsa
| Probe | Detection rate low targetb | CT perfect- matchc | CT mismatchc,d | CT delaye | Underquantitation (log10 copies/reaction)f |
|---|---|---|---|---|---|
| HP20 | 6/8 | 15.0 | 17.5 | 2.5 | 0.7 |
| HP20:QO12 (1:1) | 8/8 | 14.7 | 17.6 | 2.9 | 0.8 |
| HP20:QO12 (1:3) | 7/8 | 14.6 | 17.6 | 3.0 | 0.9 |
| HP20:QO16 (1:1) | 7/8 | 15.2 | 18.5 | 3.3 | 0.9 |
| HP20:QO16 (1:3) | 7/8 | 16.0 | 21.1 | 5.1 | 1.4 |
| HP31 | 6/8 | 16.2 | 16.4 | 0.2 | 0.1 |
| HP31:QO16 (1:1) | 8/8 | 15.2 | 15.3 | 0.1 | 0.0 |
| HP31:QO16 (1:3) | 8/8 | 15.3 | 16.3 | 1.0 | 0.3 |
aFluorescent signals in real-time RT-PCR were detected at 35°C. HP probe concentration was 200 nM.
bEight replicates of low level perfect-match targets, at 4 copies/reaction, were tested.
cCT was calculated as the median of three replicates tested. Both perfect-match and single-mismatch targets were tested at the same level, around 4 × 104 copies/reaction.
dTarget contained a single mismatch at the 12th position starting from the 5′ end of HP.
eCT Delay was determined as difference in CT between perfect-match and 1 mismatch targets (1 mismatch minus perfect-match).
fUnderquantitation was determined as difference in quantitation between perfect-match and 1 mismatch targets (prefect-match minus 1 mismatch). Quantitation was calculated off of a calibration curve established in the same experiment for each probe condition.