RNA was extracted with the RNase kit (MagMax™-96 Viral RNA Isolation Kit) using the protocol supplied by Ambion Inc. on automatic device Thermo Fisher mL (Thermo Scientific). The extracted RNAs were collected and transferred into sterile Eppendorf tubes as viral RNA extracts and preserved at −20°C until used.
The final amplification process was performed in Thermo Cycler (Applied Biosystems 7500 Fast rPCR system, Foster City, California, USA) in Micro AMP Fast Optical 96-well reaction plate. The components of master mix per 20 µl for this reaction was (for L gene), Molecular grade water (i.e., RNAse-free) 2.75 µl, Qiagen QuantiFast Probe RT PCR buffer mix (w/o ROX) 12.5 µl, 68 µl ROX to 1.7 ml QuantiFast Probe RT PCR buffer mix (400 rxn kit) 0.5 µl, NDF (12.5 µM in molecular grade water) 1 µl, NDR (12.5 µM in molecular grade water) 1 µl, LproMGB (5 µM in TE buffer) 1 µl, LproMGB 2 (5 µM in TE buffer) 1 µl, Qiagen QuantiFast RT PCR enzyme mix 0.25 µl. The cycling conditions for this test were as follows: Stage 1 - 50°C for 10 min, Stage 2 - 95°C for 5 min, Stage 3 (Step 1) - 95°C for 10 s, Stage 3 (Step 2) - 50°C for 30 s, and Stage 4 - 72°C for 30 s where steps 1 and 2 in Stage 3 were run for 40 cycles. Fluorescence was read at the end of the 2nd step of Stage 3, annealing step.
The final amplification process was performed in Thermo Cycler (Applied Biosystems 7500 Fast rPCR system, Foster City, California, USA) in Micro AMP Fast Optical 96-well reaction plate. The components of master mix per 20 µl for this reaction was (for L gene), Molecular grade water (i.e., RNAse-free) 2.75 µl, Qiagen QuantiFast Probe RT PCR buffer mix (w/o ROX) 12.5 µl, 68 µl ROX to 1.7 ml QuantiFast Probe RT PCR buffer mix (400 rxn kit) 0.5 µl, NDF (12.5 µM in molecular grade water) 1 µl, NDR (12.5 µM in molecular grade water) 1 µl, LproMGB (5 µM in TE buffer) 1 µl, LproMGB 2 (5 µM in TE buffer) 1 µl, Qiagen QuantiFast RT PCR enzyme mix 0.25 µl. The cycling conditions for this test were as follows: Stage 1 - 50°C for 10 min, Stage 2 - 95°C for 5 min, Stage 3 (Step 1) - 95°C for 10 s, Stage 3 (Step 2) - 50°C for 30 s, and Stage 4 - 72°C for 30 s where steps 1 and 2 in Stage 3 were run for 40 cycles. Fluorescence was read at the end of the 2nd step of Stage 3, annealing step.