Read more newsPCR protocol
| Stage 1 | Stage 2 | ||||
|---|---|---|---|---|---|
| Cycles | Temperature (°C) | Time (seconds) | Cycles | Temperature (°C) | Time (seconds) |
| 1 | 95 | 600 | 40 | 95 57 72 |
10 35* 20 |
* = Data collection step i.e. optics ON
- For some instruments it may be necessary to increase the annealing step to 50 seconds in order to achieve a robust
IAC signal.
Channel choices
| Instrument manufacturer | Target | IAC |
|---|---|---|
| Agilent Technologies / Stratagene | FAM | HEX |
| BioRad | FAM | HEX |
| Qiagen | Green | Yellow |
| Roche | 510nm | 580nm |
- If given the option, set the Reference Dye to None.
- For some instruments it may be necessary to calibrate (colour compensate) before use to prevent cross-talk
between channels.
Data analysis and interpretation
- Thresholds should be set to appropriate levels which are valid between runs.
- A Limit of Blank should be established using DNA samples that are free of Aspergillus in order to determine a
cut-off between positive and negative samples. - For negative reactions, a viable IAC should also be reported in order to avoid false-negative results. This is normally considered to be within ± 3 standard deviations from the average.