under the following conditions: initial denaturation temperature of 95 °C for 5 min, followed by 40 cycles of
denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at
72 °C for 1 min, and a final extension at 72 °C for 5 min. For nPCR, 2 μl
of the primary PCR product were added to another mixture with the
same reagent composition and amplified under the same conditions