Table 2. Programs of real-time PCR thermocycling condition after serial optimization.

Gene	Initial dentauration °C/time	Denaturation	Annealing	Extension	Number of cycle	Final extention
bla HSV	95°C/5 minute	94/ °C 40 sec	59/ °C 40 sec	72/ °C 40 sec	35	72/ °C 40 sec
Bla TEM	95°C/5 minute	94/ °C 40 sec	59/ °C 40 sec	72/ °C 40 sec	30	72/ °C 40 sec

A negative control was included in each run to access specificity of primers and possible contamination. The threshold cycle (Ct) was determined as the PCR cycle at which increased reporter fluorescence above the baseline could be detected.

All samples were run on PCR machine, PCR reaction data were plotted on graphic presentation by Micro software PCR program.