PCR conditions for the second round of
amplification consisted of initial denaturation at 95°C for
2 minutes, 25 cycles of 94°C for 15 seconds, 60°C for 30 sec-onds, and 70°C for 30 minutes, followed by a final exten-sion at 72°C for 6 minutes. A 5-μL aliquot of nested PCR
product was electrophoresed on a 1.5% agarose gel and
visualized using ultraviolet light after staining with 0.5μg/mL
ethidium bromide