Superscript 3 one step rt pcr system with platinum taq

Total RNA was extracted from S. latifolia leaves by using the ISOGEN reagent (FUJIFILM Wako Chemicals, Osaka, Japan) or TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). One-step RT-PCR was performed by using the SuperScript III One-Step RT-PCR System with Platinum Taq (Thermo Fisher Scientific). The primer set R2ALS1363(+) and R2ALS1551(−) was used for detecting ALSV (Figure 1). The primer set ALSR2-1213(+) and ALSR2-1484(−), which was designed to flank the multiple cloning site of the ALSV vector, was used for detecting ALSV in inoculated plants, and for checking whether the infected vector retained the insert. The reaction volume was 10 µL, and the thermal cycling conditions were 50 °C for 30 min for reverse transcription, 94 °C for 2 min to activate the DNA polymerase; followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and extension at 68 °C for 1 min; with a final extension at 68 °C for 7 min.

RNA was extracted from ICF with QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RT-PCR was carried out using JEV-specific sense primer (JE-NS3-1S; 5′-AGAGCGGGGAAAAAGGTCAT-3′) and anti-sense primer (JE-NS3-4R; 5′-TTTCACGCTCTTTCTACAGT-3′) [20 (link)] with SuperScript III One-Step RT-PCR System with Platinum Taq (Thermo Fisher Scientific Inc, Waltham, MA). RT-PCR was performed 1 cycle at 53 °C for 30 min and 94 °C for 2 min; 40 cycles at 94 °C for 15 s, 53 °C for 30 s, and 68 °C for 90 s; and 1 cycle at 68 °C for 5 min.

RT-PCR tests were carried out using the SuperScript III One-Step RT-PCR System with Platinum^® Taq (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions except that the total reaction volume was 25 μL. The PCR products were analysed by electrophoresis in 1.5% agarose gels that were stained with SYBR^® Safe DNA gel stain (Invitrogen) for visualization.
For preliminary screening by RT-PCR the total RNA extract of each sample was tested for ApMV and PDV using previously published primers that were specific for RNA3 of each virus [25 (link),26 (link)].
Total RNA extracts from the Prunus tree samples that tested positive for the RNA3 for ApMV and/or PDV were used for RT-PCR amplification of partial segments of the (MT) gene on RNA1, RdRp gene on RNA2, and CP gene on RNA3 of both viruses using primers developed for this study (Table 2). Cycling conditions consisted of: a reverse transcription step at 48 °C for 45 min; denaturation at 94 °C for 2 min, followed by 35 cycles, denaturing at 94 °C for 30 s, annealing for 30 s at the appropriate temperature for each primer pair (Table 2), elongation at 72 °C for 1 min; and a final elongation step at 72 °C for 10 min.