Abstract
Mosquito-borne diseases pose ongoing global health concerns, demanding more cost-efficient methods to detect pathogens to support enhanced surveillance efforts. This study introduces an adapted TRIzol-based high-throughput RNA extraction protocol, tailored for the detection of California serogroup viruses in pooled mosquito samples in a rapid and cost-effective manner. This approach provided consistent RNA yields and sensitive viral detection relative to two commercial extraction kits (QIAGEN RNeasy Mini Kit and MACHEREY-NAGEL NucleoSpin RNA Plus Kit). The incorporation of a user-friendly and non-spiking-based RT-qPCR internal control designed for the 18S rRNA gene in mosquitoes minimizes potential false positives/negatives, improving the fidelity of viral detection outcomes. Effective RNA yields, purity, and successful target amplification across 25 mosquito species and varied pool sizes (1-50 mosquitoes per tube) affirm the reliability of our approach. The extraction method is cost-effective, with an incurred cost of $0.58 CAD per sample, in contrast to the $5.25 CAD cost per sample of the two kits, rendering it promising for mosquito-borne disease surveillance initiatives.