Superscript 3 one step

Viral RNA was extracted from culture supernatant using the Macherey-Nagel (Düren, Germany) Viral RNA isolation kit as per the manufacturer’s instructions. RT-PCR was performed using Invitrogen Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase and a generic mesonivirus primer set NidoF1: 5′-GTTGTATGCTATGCCGYCG-3′, NidoR1: 5′-TCCATAGTATCGTAGCAATTCC-3′ and the following cycling conditions; reverse transcription: 45 °C/30 min; PCR: one cycle 94 °C/2 min; 40 cycles 94 °C/30 s, 45 °C/30 s, 68 °C/1 min; followed by a final extension cycle of 68 °C/5 min. PCR products were confirmed by excising under UV and DNA extracted using Macherey-Nagel NucleoSpin Gel and PCR Clean-up as per manufacturer’s instructions. Samples were submitted for Sanger sequencing at AGRF.

RV was detected by RT-PCR to amplify the conserved regions on the VP7 and VP4 genes using SuperScript III One-step RT-PCR system with Platinum Taq (Invitrogen, Carlsbad, CA) as previously described [28 (link)]. The VP7 gene was amplified by Beg9 and End9 primers, while Con2 and Con3 primers were used to amplify the VP4 gene.

RT-PCR assays were performed using the RNA obtained from leaf samples as templates (diluted to 50 ng/µL) with a SuperScript III One-Step (Invitrogen, Carlsbad, CA, USA) commercial system, following the manufacturer’s instructions. Following primers were used to amplify a 688 bp fragment corresponding to the p25 (CP gene) [15 ]: R731-7 (5′-CGGAACGCAACAGATCAACG-3′) and RF-137 (5′-ATTATGGACGACGAAACAAA-3′). It was observed previously that severe CTV isolates contained an EcoRI site in the 5′ end of the CP gene. In order to determine the severity of the CTV strain, the RT-PCR products were digested with EcoRI and analyzed by gel electrophoresis. These products were cloned in pDrive (Qiagen, Hilden, Germany) and sequenced to confirm the presence or absence of this polymorphism.