Amplification and data acquisition are carried out in a MiniOpticon® (Bio-Rad) real-time PCR machine using the following program:
- Initial enzyme activation step:
a. 98°C for 15 min
- Followed by 40 cycles of:
a. denaturation at 98°C for 5 sec
b. annealing/extension at 63°C for 10 sec
c. melt curve analysis from 65-95°C (in 0.5°C increments) 10 sec/step
Specificity and the absence of non-specific amplification are determined based on the melting temperature (Tm) of the amplified products (see also molecular methods paper of the BEEBOOK (Evans et al., 2013)).