{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T15:07:27Z","timestamp":1774278447607,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,20]]},"group-title":"Microbiology","reference-count":45,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T00:00:00Z","timestamp":1773964800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The co-circulation and rapid expansion of the genus\n                  <jats:italic>Orthoflavivirus<\/jats:italic>\n                  , including dengue virus (DENV), Zika (ZIKV), and Japanese encephalitis virus (JEV), pose significant global health challenges. Developing inclusive pan-genus molecular diagnostics is hindered by high nucleotide divergence (&gt;25%\u201330%) and the computational limitations of traditional multiple sequence alignment in detecting conserved motifs across large datasets. To overcome these limitations, we developed a systematic alignment-free design pipeline that uses rigorous k-mer analysis and compacted De Bruijn graphs. We analyzed 11,846 RefSeq viral genomes to identify phylogenetically conserved, functionally relevant signatures within the\n                  <jats:italic>Orthoflavivirus<\/jats:italic>\n                  genus as a case study. The pipeline identified a conserved 600-bp region within the non-structural protein 5 gene, facilitating the design of a broad-spectrum TaqMan RT-qPCR assay. Analytical validation against standard reference strains demonstrated a limit of detection of 1\u201310 copies\/\u00b5L for DENV1\u20134, ZIKV, and JEV, with no cross-reactivity against non-target pathogens. In a clinical evaluation of archived samples, the assay achieved 97.33% overall accuracy. It demonstrated 100% sensitivity and specificity for DENV serotypes, yielding significantly earlier cycle threshold (Ct) values compared to a standard commercial kit, while ZIKV detection showed 100% specificity with 71.43% sensitivity. This study validates an alignment-free, k-mer guided approach for uncovering conserved diagnostic targets in highly variable viral genera. The resulting assay offers a robust tool for frontline surveillance, and the computational framework provides a scalable solution for future pandemic preparedness.\n                <\/jats:p>","DOI":"10.64898\/2026.03.17.712358","type":"posted-content","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T16:50:19Z","timestamp":1774025419000},"source":"Crossref","is-referenced-by-count":0,"title":["Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay"],"prefix":"10.64898","author":[{"given":"Khanate","family":"Sayasit","sequence":"first","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chutikarn","family":"Chaimayo","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 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