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Conventional detection methods exhibit critical limitations in multiplexing capability, prolonged turnaround time, and procedural complexity. These factors hinder rapid outbreak response and underscore the necessity for improved detection platforms. This study developed a quadruplex quantitative real\u2010time PCR (qRT\u2010PCR) assay utilizing Minor Groove Binder (MGB) probes for the simultaneous detection of JEV, WNV, DENV, and ZIKV. The MGB probe\u2010based qPCR assay demonstrated high specificity in distinguishing these four orthoflaviviruses and exhibited significantly enhanced sensitivity compared to conventional TaqMan probes, achieving detection limit of 2 copies\/reaction. This method was successfully applied to detect virus\u2010positive samples in cell cultures, laboratory mosquitoes, and field\u2010collected mosquitoes, thereby validating the practical utility of the MGB probe\u2010based qPCR assay. Compared to conventional multiplex detection methods, our MGB\u2010based approach provides a promising detection platform, thereby demonstrating its potential utility in the high\u2010throughput surveillance of mosquito\u2010borne orthoflaviviruses.<\/jats:p>","DOI":"10.1002\/jmv.71049","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T16:30:50Z","timestamp":1782923450000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Application of a Quadruplex TaqMan\u2010MGB qPCR Assay for Simultaneous Detection of Important Mosquito\u2010Borne Orthoflaviviruses"],"prefix":"10.1002","volume":"98","author":[{"given":"Xinyao","family":"Li","sequence":"first","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine Huazhong Agricultural University Wuhan Hubei China"},{"name":"Hubei Hongshan Laboratory Wuhan Hubei 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