Iq multiplex powermix kit

All real-time PCR reactions were conducted with Bio-Rad CFX96™ Touch™ Real-time PCR Detection System. For DNA targets, iQ™ Multiplex Powermix kit (Bio-Rad, Hercules, CA, USA) was used. A volume of 20 μL PCR reaction contains 10 μL 2 × IQ Powermix, 0.8 μL primer mix (final concentration of 400 nM), 0.4 μL probe mix (final concentration of 200 nM), 1 μL DNA template, and 7.8 μL nuclease-free water. The reaction condition involved a 95 °C incubation for 5 min, followed by 45 cycles of denaturation at 95 °C for 15 s and a combined annealing and extension step at 60 °C for 45 s. Singular or multiplex real-time RT-PCR for RNA targets were carried out with Path-ID Multiplex One-Step RT-PCR Kit (Applied Biosystems/Life Technologies, Grand Island, NY, USA). Each reaction contains 10 μL 2 × Multiplex RT-PCR Buffer, 1 μL Multiplex Enzyme Mix, 0.8 μL primer mix (final concentration of 400 nM), 0.4 μL probe mix (final concentration of 200 nM), 1 μL RNA and 6.8 μL nuclease-free water. The reaction condition involved a reverse transcription step at 48 °C for 10 min and RT inactivation and denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 45s. If a subpanel contained both DNA and RNA viruses, the real-time RT-PCR protocol was used. The final results were analyzed using Bio-Rad CFX Manager 3.0 software.