All four primers (PNRNA1-F, PNRNA2-F, PNRNA3-F, and PNRNA123-R) were utilized in a single tube to amplify all three RNA fragments of PNRSV concurrently in one and two-step mRT-PCR. In one-step mRT-PCR, a total of 10 µl of reaction was set up in a single PCR tube using total plant RNA (400 ng), three forward (0.25 uM each), and one reverse (0.75 uM) primer together with all of the components of the OneTaq One-Step RT-PCR Kit (New England Biolabs, USA) in a thermocycler 8800 (Agilent Technologies, USA). Cycling conditions were identical to those employed in one-step RT-PCR.
In two-step mRT-PCR, 10 µl reaction containing 1 µl cDNA, 5 µl master mix (2×) of ProtoScript AMV LongAmpTaq RT-PCR Kit (New England Biolabs, USA), 0.25 µl (from 10 pM) each three forward, 0.75 µl (from 10 pM) of reverse primer and 2.5 µl of nuclease-free water, was set up in a single PCR tube in a thermocycler 8800 (Agilent Technologies, USA). The PCR amplification was performed with a 1 min denaturation step at 95 °C followed by 35 cycles of 95 °C for 30 s, 51 °C for 30 s, 68 °C for 3.20 min and a final extension at 68 °C for 10 min.
In two-step mRT-PCR, 10 µl reaction containing 1 µl cDNA, 5 µl master mix (2×) of ProtoScript AMV LongAmpTaq RT-PCR Kit (New England Biolabs, USA), 0.25 µl (from 10 pM) each three forward, 0.75 µl (from 10 pM) of reverse primer and 2.5 µl of nuclease-free water, was set up in a single PCR tube in a thermocycler 8800 (Agilent Technologies, USA). The PCR amplification was performed with a 1 min denaturation step at 95 °C followed by 35 cycles of 95 °C for 30 s, 51 °C for 30 s, 68 °C for 3.20 min and a final extension at 68 °C for 10 min.