Quantifast rt mix

To evaluate the sanitary status of the 25 grapevines, further assays were performed by real-time RT-PCR according to the methods developed by Bianchi et al. (2010 ). One-step multiplex real-time RT-PCR was used for the detection of GVA, GFLV, ArMV, GLRaV-1, and GLRaV-3. Five microliters of RNA was added to 12.5 μl of 2X QuantiFast multiplex RT-PCR Master mix without ROX (Qiagen, Hilden, Germany) supplemented with 0.25 μl of QuantiFast RT Mix (Qiagen, Hilden, Germany), 0.4 μM final concentration of each primer and 0.2 μM of the probes, and RNase-free water to a final volume of 25 μl. Multiplex one-step real-time RT-PCR was performed on a CFX96 real-time system (Bio-Rad, Hercules, CA, USA) using the following amplification conditions: 50 °C for 30 min, 95 °C for 5 min followed by 45 cycles of 95 °C for 5 s, and 60 °C for 30 s. All samples were analyzed at least twice and each run included a no template control, a negative control, and a positive control for each virus.
All real-time PCR data were analyzed using the CFX Manager software 2.0 (Bio-Rad, Hercules, CA, USA). Samples were considered positive for a mean Ct value < 30, with a baseline threshold set to 100 RFU in all PCR reactions (Bianchi et al. 2010 ).

On the day of collection, harvested serum (0.05 mL) was added to TRIzol^® LS (5X volume; i.e., 0.25 mL) and stored at ≤−65 °C until RNA exaction. RNA was extracted from samples using the Zymo Research Direct-zol^™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). For sample quantification, each assay plate contained a standard curve prepared using MARV VP40 gene synthetic RNA (1.0 × 10³ to 1.0 × 10¹⁰ genome equivalents/µL [GEq/µL] in duplicate wells). For the qRT-PCR, QuantiFast Probe RT-PCR Master Mix and QuantiFast RT Mix (Qiagen, Ilden, Germany) were used in conjunction with Forward primer: 5′- CCAgTTCCAgCAATTACAATACATACA-3′, Reverse primer: 5′- gCACCgTggTCAgCATAAggA-3′ and Probe: 5′-6FAM- CAATACCTTAACCCCC-MGBNFQ-3′. Primers and probe targeted the VP40 gene from MARV (GenBank accession no. DQ447660). The qRT-PCR was conducted on a Bio-Rad CFX96^TM Real-Time PCR.

50 µL of sera was added to 250 µL of TRIzol LS (Invitrogen, Carlsbad CA, USA) and stored at ≤−70 °C until RNA extraction using a Zymo Research Direct-zol™ RNA MiniPrep kit (Zymo Research, Irvine CA, USA). Final elutions (containing purified RNA) were stored at ≤−70 °C until use. For sample quantification, each assay plate included a standard curve prepared using an HPLC-purified synthetic EBOV RNA standard containing the conserved EBOV glycoprotein sequence (GenBank accession no. AY729654) ranging from 1.0 × 10³ to 1.0 × 10¹⁰ genome equivalents/µL (GEq/μL]. Samples were run in duplicate wells. For the RT-PCR, QuantiFast Probe RT-PCR Master Mix (Qiagen, Germantown MD, USA), and QuantiFast RT Mix (Qiagen, Germantown MD, USA) were used in conjunction with Forward primer: 5′- ggA Tgg AgC TTT CTT CCT CTA Tg -3′, Reverse primer: 5′- TAC CCC CTC AgC AAA ATT gAC T -3′, and Probe: 5′-6FAM- CAg gCT ggC TTC AAC TgT AAT TTA CAg Agg -MGBNFQ-3′. The RT-PCR was performed using a Bio-Rad CFX96TM Real-Time PCR detection system.