Real-Time RT-qPCR for ARPV and ZIKV

Viral RNA was extracted using QIAmp Viral RNA Mini kits (QIAGEN) according to the manufacturer’s instructions. RT-qPCR was performed using iTaq^TM Universal Probes One-Step kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s guidelines. ARPV primers ARPV-7562F (5′-CGGTGTTCATTGAGGATGAC-3′), ARPV-7714R (5′-TGATACGTCCAGGTTCGGTA-3′) and probe TR9096-P2-7680F (5′-6FAM-CGCTGCCTCATGGCAATTCG-BHQ1-3′) were used for the detection and quantification of ARPV and ARPV/ZIKV. In vitro transcribed RNA was used to generate standard curves. Primers ZIKV-1086F (5′-CCGCTGCCCAACACAAG-3′), ZIKV-1162cR (5′-CCACTAACGTTCTTTTGCAGACAT-3′) and probe ZIKV-1107-FAM (5′-6FAM-AGCCTACCTTGACAAGCAGTCAGACACTCAA-BHQ1-3′) were used for the detection and quantification of ZIKV as previously described [35 (link)].