Quantitative real time PCR (qPCR)

The qPCR assay was performed in the CFX96 Real-Time System (Bio-Rad) in a final volume of 20 μl reaction that contained 10 μl of SsoAdvanced™ Universal Probes Supermix (Bio-Rad), 900 nM forward and reverse primers and 250 nM probe, 2 μl of S. citri DNA and final volume made up with double distilled water. The thermal cycling conditions consisted of initial denaturation at 95°C for 5 min, then 40 cycles of denaturation at 95°C for 10 s, and annealing at 57°C for 30 s. We have performed the qPCR with primer/probe concentration of 300nm/150nm and 900nm/250nm and obtained no significant Cq value changes. We used the same concentration of primer/probe for ddPCR as well, to maintain the same reaction conditions. Each run included S. citri positive and negative controls (DNA from healthy Navel sweet orange leaves and columella) along with a no template control (NTC). A standard curve was constructed for serial dilutions of S. citri plasmid DNA and included in all runs to produce quantitative results.