Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada.
Sci Rep. 2024 Jan 29;14(1):2339. doi: 10.1038/s41598-024-52534-1.
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.
蚊媒传染病持续对全球健康构成威胁，需要更具成本效益的方法来检测病原体，以支持加强监测工作。本研究介绍了一种改良的 TRIzol 基高通量 RNA 提取方案，旨在快速、经济高效地检测混合蚊虫样本中的加利福尼亚血清群病毒。与两种商业提取试剂盒（QIAGEN RNeasy Mini Kit 和 MACHEREY-NAGEL NucleoSpin RNA Plus Kit）相比，该方法提供了一致的 RNA 产量和灵敏的病毒检测。引入一种针对蚊虫 18S rRNA 基因的基于用户友好且不添加 Spike 的 RT-qPCR 内参，可最大程度地减少潜在的假阳性/假阴性，提高病毒检测结果的准确性。我们的方法在 25 种蚊虫物种和不同的混合样本量（每管 1-50 只蚊虫）中均能有效提取 RNA、保证纯度并成功扩增靶标，证实了其可靠性。与每个样本 5.25 加元的两种试剂盒相比，该提取方法的成本效益高，每个样本的成本仅为 0.58 加元，有望用于蚊媒传染病监测计划。
