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; Department of Pharmacology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok, Thailand; Department of Bioprocess Technology, Faculty of Technology, Rajarata University of Sri Lanka, Mihintale, Sri Lanka; Industrial Sensor Technology Research Team, National Center for Genetic Engineering and Biotechnology, National Sciences and Technology Development Agency at King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok, Thailand; Sensor Technology Laboratory, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok, Thailand; Nanoscience & Nanotechnology Graduate Programme, Faculty of Science, King Mongkut's University of Technology Thonburi, Bang Mot, Bangkok, Thailand
Type
Article
Source Title
PeerJ
ISSN
21678359
Year
2025
Volume
13
Open Access
All Open Access; Gold Open Access; Green Open Access
Publisher
PeerJ Inc.
DOI
10.7717/peerj.19820
Abstract
Background. The genus Orthoflavivirus 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. Methods. Several previously published broad-specific primers targeting the genus Orthoflavivirus 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. Results. The primer FlaviPath-F and FlaviPath-R targeted 36% of the selected pathogenic orthoflavivirus species without cross-reacting with non-pathogenic strains based on in silico 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 104 RNA copies. Discussion. 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. Copyright 2025 Thayanukul et al.
