One The following RT-PCR cycling conditions were used: 60uC for 5 min, 4uC for 10 min, 55uC for 45 min, 95uC for 10 min, followed by 8 cycles of 95uC for 30 seconds, 48uC for 30 seconds, and 72uC for 30 seconds, with the 48uC annealing temperature increasing 0.9uC each cycle. The PCR was then continued for 37 additional cycles of 95uC for 15 seconds, 56uC for 20 seconds, and 72uC for 20 seconds. The RT-PCR cycle ended with a final extension of 2 min at 72uC followed by a 4uC hold.