qPCR primer–probe sets allow the specific monitoring of genomic and subgenomic challenge RNA replication.
(A) The GFP primer–probe set targets the 162–212 region of the GFP gene, while the genomic GFP forward primer targets the last 27 bases of nsP4, the probe the 31–46 position of the subgenomic promoter, and the reverse primer the first 18 bases of GFP. (B) The mCherry primer–probe set targets the 161–237 region of the mCherry gene. (C,D) One million plaque forming unit (PFU) of CHIKV-GFP, CHIKV-mCherry or SINV-GFP were lysed. RNA was subsequently extracted and RT–qPCR was performed using the indicated primer–probe sets.