Thayanukul Parinda, Morales Vargas Ronald Enrique, Sujijun Konkamon, Khumpeera Pimchanok, Suksawat Kittiya, Wijegunawardana Nahallage Dona Asha Dilrukshi, Rijiravanich Patsamon, Surareungchai Werasak, Kittayapong Pattamaporn
Department of Biology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok, Thailand.
Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University, Salaya, Nakhon Pathom, Thailand.
PeerJ. 2025 Aug 26;13:e19820. doi: 10.7717/peerj.19820. eCollection 2025.
The genus primarily consists of arthropod-borne viruses capable of infecting vertebrate hosts and causing serious human diseases such as dengue fever, Zika fever, Japanese encephalitis, West Nile fever, and yellow fever. This study describes the development of a simple and field-deployable detection system for multiple pathogenic orthoflavivirus species using the recombinase polymerase amplification (RPA) technique.
Several previously published broad-specific primers targeting the genus were evaluated. A new primer pair, FlaviPath-F and FlaviPath-R, was designed and tested for its applicability in an RPA assay. The RPA protocol was experimentally optimized, with a focus on determining the assay's sensitivity and assessing the primers' specificity against pathogenic orthoflaviviruses.
The primer FlaviPath-F and FlaviPath-R targeted 36% of the selected pathogenic orthoflavivirus species without cross-reacting with non-pathogenic strains based on analysis. The RPA assay successfully amplified DNA oligonucleotides from dengue virus, Japanese encephalitis virus, Zika virus, and West Nile virus. Furthermore, positive amplification was observed in RNA samples extracted from mosquitoes infected with dengue and Zika viruses. The RPA assay demonstrated high sensitivity, with the potential to detect as few as a single viral RNA copy, although confirmation is needed for concentrations below the detection limit of 10 RNA copies.
This is the first study to develop an RPA-based method for the detection of multiple orthoflavivirus pathogens in mosquito vectors. The reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (RT-RPA-LFD) platform offers a rapid, cost-effective tool for identifying regions at risk of arboviral transmission, supporting the targeting of individual viral diseases. This technique holds promise as an early warning system for emerging arboviral threats in public health.
该属主要由节肢动物传播病毒组成，这些病毒能够感染脊椎动物宿主并引发严重的人类疾病，如登革热、寨卡热、日本脑炎、西尼罗河热和黄热病。本研究描述了一种使用重组酶聚合酶扩增（RPA）技术开发的用于多种致病性正黄病毒属物种的简单且可现场部署的检测系统。
对先前发表的几种针对该属的广泛特异性引物进行了评估。设计了一对新引物FlaviPath-F和FlaviPath-R，并测试了其在RPA检测中的适用性。通过实验对RPA方案进行了优化，重点是确定检测的灵敏度并评估引物对致病性正黄病毒的特异性。
基于分析，引物FlaviPath-F和FlaviPath-R靶向36%的选定致病性正黄病毒属物种，且不与非致病性菌株发生交叉反应。RPA检测成功扩增了来自登革病毒、日本脑炎病毒、寨卡病毒和西尼罗河病毒的DNA寡核苷酸。此外，在从感染登革热和寨卡病毒的蚊子中提取的RNA样本中观察到阳性扩增。RPA检测显示出高灵敏度，有可能检测到低至单个病毒RNA拷贝，尽管对于低于10个RNA拷贝检测限的浓度还需要进一步确认。
这是第一项开发基于RPA方法用于检测蚊媒中多种正黄病毒病原体的研究。带有侧向流动试纸条的逆转录重组酶聚合酶扩增检测（RT-RPA-LFD）平台为识别虫媒病毒传播风险区域提供了一种快速、经济高效的工具，有助于针对个别病毒性疾病。这项技术有望成为公共卫生领域新兴虫媒病毒威胁的早期预警系统。
