Real-time qPCR for DCH Quantification

Real-time qPCR for DCH was performed as described previously [7 (link)]. For absolute DCH DNA quantification, a 1.4 kb-long fragment of the polymerase region of the Australian reference strain AUS/2016/Sydney was cloned using the TOPO XL-2 PCR cloning kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Tenfold dilutions at 10⁰–10⁹ copies of plasmid per reaction were used to generate the standard curve. Standards and samples were run in triplicate. Ultrapure water and salmon sperm DNA were used as negative controls. Reactions were 25 μL, comprising 1 μL of neat template DNA plus 9 μL of water; or 10 μL plasmid standard and 15 μL of master mix, comprising IQ Supermix (Bio-Rad Laboratories SRL, Segrate, Italy), containing 0.6 μmol/L of each primer and 0.1 μmol/L of probe. Thermal cycling consisted of activation of Taq DNA polymerase at 95 °C for 3 min and 42 cycles of denaturation at 95 °C for 10 s and annealing-extension at 60 °C for 30 s. A sample was defined as positive if ≥10 copies of DCH DNA were detected in at least ≥ two of three replicates with a Ct value of ≥38.5. Thus, our PCR assay had a lower limit of quantification of 2500 copies/mL of blood. The cut-off for R-squared was 0.980 and for efficiency was 90–110%.