CHIKV Detection in Human Plasma and Mosquitoes

RNA was extracted from human plasma samples (140 µL) using the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Mosquitoes were pooled per 50 individuals or less and homogenized with a vortex in ZR bashing bead lysis tubes containing 1 mL DNA/RNA shield. RNA was subsequently extracted from 200 µL homogenate according to the protocol of the Quick-DNA/RNA^TM Pathogen Miniprep Kit (Zymo Research, Germany). RNA from phocine distemper virus (PDV) was added to all samples as an internal RNA extraction and PCR inhibition control [16 (link)]. A CHIKV-specific RT-qPCR was then performed with 5 µL RNA in a 25 µL reaction using the iTaq Universal Probes One-Step Kit from Bio-Rad by amplifying a 77 bp part of the nonstructural protein 1 (NSP-1) gene with primers and probes (S Table 1) detecting the African and Asian CHIKV strains as previously described [17 (link)]. Cycling conditions were 10 min at 50°C, a denaturation step of 5 min at 95°C, followed by 50 cycles of 10 s at 95°C and 30 s at 60°C. A PDV RT-qPCR was run in parallel (S table 1). RNA samples with Ct-value >30 were concentrated to 10 µL using the Zymo RNA Clean & Concentrator^TM 5 kit (Zymo Research) prior to sequencing.