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2, 1x PCR buffer (20 mM Tris-HCl, pH 8.3, 500 mM KCl), 0.2 mM dNTP mix, 0.3 μM of each N-gene primer, 2.5 U of Taq DNA polymerase, and 4 μL of cDNA. The amplification began with denaturation at 94°C for 3 min, followed by 35 cycles of denaturation for 15 s at 94°C, annealing for 30 s at 60°C, extension for 30 s at 72°C, and followed by a final elongation period of 5 min at 72°C.
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