Multiplex RT-qPCR for Detecting Arboviruses

To set a RT-qPCR assay for a single multiplex reaction capable of simultaneously detecting three viruses, we used sets of primers and probes employed for DENV [27 (link)], ZIKV [28 (link)], and CHIKV [29 (link)] detection (Table S1). Reactions were performed using the QuantiNova Probe RT-PCR kit (Qiagen, catalog #208354, Hilden, Germany). The mixture consisted of 0.08 µL of each primer (800 nM), 0.04 µL of each probe (100 nM), 5.0 µL of QuantiNova Probe RT-PCR Master Mix (5×), 0.1 µL of QuantiNova Probe RT Mix, 0.05 µL of ROX passive reference dye, and 3.5 µL of the transcripts dilutions, in a final volume of 10 μL. Cycling conditions were 15 min at 45 °C and 5 min at 95 °C, followed by 45 cycles of 5 s at 95 °C and 45 sec at 60 °C. Multiplex RT-qPCR assays were done on a QuantStudio 5 Real-Time PCR System (Applied BioSystems, Waltham, MA, USA), with automatic baseline and threshold. The singleplex reaction (separate for each virus) was carried out using the same PCR conditions and concentrations from the multiplex reaction, only adjusting the water volume.