Abi prism 7500 thermocycler

The virus titer was quantified in the inoculated plants using SYBR Green-based quantitative real-time PCR (qPCR) with begomovirus/betasatellite-specific primers (Supplementary Table S1). The qPCR assays were carried out in triplicate in a 20-µl reaction volume containing 10 µl of SYBR Green dye (2×), 0.2 µM primers, and 100 ng of template DNA. The qPCR was carried out using an ABI Prism 7500 Thermocycler (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95°C for 10 min followed by 40 cycles each of denaturation at 95°C for 1 min; annealing at 60°C for 30 s; and extension at 72°C for 1 min. Individual standard curves for BYVMV and BYVB full-length clones were prepared by making a 100-fold serial dilution from 10 9 to 10 2 copies (Supplementary Fig. S1). The absolute quantification of viral molecules was performed as described by Shafiq et al. (2017) .