Real-time PCR was carried out in a 12 ml final volume consisting of 6 ml IQ SYBR Green Supermix (Bio-Rad), 0.34 ml each primer (10 mM),2 ml template DNA and water. PCRs were performed in triplicate on all samples. PCR was performed on anMJ Opticon thermocycler (Bio-Rad) using the following cycling protocol: initial denaturation step at 95 uC for 3 min, followed by 40 cycles at 95 uC for 20 s and 60 uC for 45 s followed by dissociation curve analysis from 65 to 95 uC.