Quantifying ZIKV in Mosquito Saliva

To compare plaque assays with quantitative reverse transcription PCR (RT-qPCR), we performed RT-qPCR on saliva samples from mosquitoes exposed to 10⁵ and 10⁶ PFU/mL at days 4 and 20 post-infection. Viral RNA was extracted from saliva samples (QIAamp Viral RNA Mini Kit, Qiagen) and reverse-transcribed to cDNA (High Capacity RNA-to-cDNA Kit, Applied Biosystems). ZIKV genome copies were measured with RT-qPCR reaction assay using TaqMan Gene Expression Master Mix (Applied Biosystems), primers (F: ZIKV 1086, R: ZIKV 1162c; Invitrogen Custom Primers) and probes (ZIKV 1107-FAM; TaqMan MGB Probe) [30 (link)]. Each sample was analyzed in duplicate, and each assay contained a standard curve (ZIKV molecular clone), no template, and no primer controls. We extrapolated ZIKV copy numbers from the generated standard curve using the Applied Biosystems protocol. The limit of detection was experimentally established to be 30 copies (10⁻¹⁶ g). Final copy numbers were adjusted by back-calculations to the total RNA and cDNA volume and expressed as copies per saliva sample.