The real-time PCR was carried out using the ABI AmpliTaq Gold DNA polymerase system with an icycler IQ Realtime PCR Detection System (Bio-Rad Corp., Hercules, CA) according to the manufacturer's instructions. The reaction, data acquisition and analysis were performed using iCycler IQ optical system software. The Real-time PCR was performed in an 25 μL reaction mixture containing 1 × PCR buffer, 0.2 mmol/L dNTPs, 1 U Taq and 1 μL DNA template according to the manufacture's instructions. Autoclaved nanopure water was added to bring the final volume to 25 μL. The two-step PCR cycling condition was as follows: initial denaturation and hot-start Taq The conventional PCR was performed and optimized on a Mycycler™ thermo cycler system (Bio-Rad Corp., Hercules, CA, USA) with a 50 μL PCR reaction system containing 1 × PCR buffer, 0.2 mmol/L dNTPs mixture, 1.25 U rTaq (Takara Bio Inc., Shiga, Japan), 0.5 μmol/L each forward and reverse primers and 1 μL template DNA. All PCR experiments were carried out in 0.2 ml thin-walled tubes with the following cycle parameters: The mixture was subjected to initial denaturation at 95°C for 1 min, followed by 50 cycles of 95°C for 60 s, annealing for 60 s, extension at 72°C for 60 s, and one cycle of final extension at 72°C for 5 min. The amplified 498 bp product then underwent electrophoresis on 1.0% agarose gels. Electrophoresis was carried out at 100 V in a Mini-sub (Bio-Rad Corp., Hercules, CA, USA) gel electrophoresis unit and gels were viewed under a UV transilluminator. The conventional PCR reactions were optimized based on MgCl 2 concentration and annealing temperature selection criteria in a similar way as that of Real-time PCR and the selection was made by the brightness of the amplified 498 bp fragments on the agarose gel under a UV transilluminator.