A gradient mRT-PCR was then conducted to determine the opti-mal annealing temperature for mRT-PCR (Fig. 2). Results showedthat annealing temperatures of 55–60◦C raised the efficient ampli-fication of these viruses. The optimized thermal profile of theRT-PCR was: 5 min at 94◦C, followed by 35 cycles of 1 min at 94◦C,1 min at 60◦C, 1.5 min at 72◦C and a final incubation at 72◦C for10 min.