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PCR 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 Aspergillus 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.