Faísca-Silva David, Seixas Gonçalo, Nunes Mónica, Parreira Ricardo
Institute of Hygiene and Tropical Medicine (IHMT), NOVA University of Lisbon, Rua da Junqueira 100, 1349-008 Lisbon, Portugal.
Global Health and Tropical Medicine (GHTM), Rua da Junqueira 100, 1349-008 Lisbon, Portugal.
Pathogens. 2024 Oct 11;13(10):892. doi: 10.3390/pathogens13100892.
Mosquitoes serve as vectors for many arthropod-borne viruses (arboviruses) that are responsible for millions of human infections and thousands of deaths each year. Among these arboviruses, O'nyong-nyong virus (ONNV) is an African alphavirus mainly transmitted by mosquitoes. ONNV can be detected through serological or molecular tests, the first showing cross-reactivity to co-circulating alphaviruses and requiring technically demanding confirmation, while the latter, usually based on real-time PCR, are costly and demand specific equipment. Isothermal amplification approaches, such as Loop-Mediated Isothermal Amplification (LAMP), should therefore provide a cost-effective, sensitive, and specific alternative for virus detection, suitable for the resource-limited regions where ONNV circulates up to the present time. Here, we describe the development and optimization of a rapid and highly sensitive (10 pfu/reaction) RT-LAMP assay for ONNV detection. Additionally, we demonstrate that it is possible to bypass the RNA extraction step, reducing sample handling time and costs. The final RT-LAMP is a promising field detection tool for ONNV, enabling a better understanding of its impact and serving as a point-of-care diagnostic method.
蚊子是许多节肢动物传播病毒（虫媒病毒）的传播媒介，这些病毒每年导致数百万人感染和数千人死亡。在这些虫媒病毒中，奥尼昂-尼昂病毒（ONNV）是一种主要由蚊子传播的非洲甲病毒。可以通过血清学或分子检测来检测ONNV，前者显示出与共同传播的甲病毒的交叉反应性，需要技术要求较高的确认，而后者通常基于实时PCR，成本高昂且需要特定设备。因此，等温扩增方法，如环介导等温扩增（LAMP），应为病毒检测提供一种经济高效、灵敏且特异的替代方法，适用于目前ONNV传播的资源有限地区。在此，我们描述了一种用于ONNV检测的快速且高度灵敏（10个空斑形成单位/反应）的逆转录环介导等温扩增（RT-LAMP）检测方法的开发和优化。此外，我们证明可以绕过RNA提取步骤，减少样本处理时间和成本。最终的RT-LAMP是一种很有前景的ONNV现场检测工具，能够更好地了解其影响，并作为一种即时诊断方法。
