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In this study, a duplex reverse transcription multi-enzyme isothermal amplification (RT-MIRA) assay was established for the simultaneous detection of DENV and CHIKV, followed by a nested RT-MIRA assay for DENV serotyping (DENV-1 to -4).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Specific primers and probes targeting the DENV 3\u2032-UTR, CHIKV E1 gene, and four DENV serotypes were designed. The duplex RT-MIRA and nested DENV RT-MIRA serotyping reaction systems were optimized at 39\u00a0\u00b0C with portable fluorescence or lateral flow dipstick readouts. For methodological validation, specificity was evaluated against 35 related pathogens, and the 95% limit of detection (LOD95) was determined via probit regression. For clinical validation, serum samples from 236 suspected patients were tested, benchmarking against RT-qPCR and serology. Statistical analyses included the Wilson score method for calculating 95% confidence intervals (\n                      <jats:italic>CIs<\/jats:italic>\n                      ) and Cohen\u2019s kappa (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      ). For external verification, 12 CHIKV-positive clinical samples and 5 artificially simulated co-infection samples were retrospectively analyzed to validate assay accuracy.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The duplex RT-MIRA assay exhibited no cross-reactivity with other pathogens. The LOD95 values were 13.47 copies\/\u03bcl for DENV and 10.49 copies\/\u03bcl for CHIKV. Clinical validation demonstrated sensitivities of 96.15% (95%\n                      <jats:italic>CI<\/jats:italic>\n                      : 89.28%\u201398.67%) for DENV and 88.89% (95%\n                      <jats:italic>CI<\/jats:italic>\n                      : 67.20%\u201396.90%) for CHIKV. Specificity was 100% (95%\n                      <jats:italic>CI<\/jats:italic>\n                      : 92.87%\u2013100%) for both. Agreement with RT-qPCR was strong for DENV (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      \u2009=\u20090.96) and CHIKV (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      \u2009=\u20090.92). The nested RT-MIRA serotyping assay showed high sensitivity (LOD95: 1.6\u201318.7 copies\/\u03bcl) without cross-reactivity, accurately differentiating 75 DENV-positive samples into 71 DENV-1 and 4 DENV-2. In the external verification, the assay accurately detected 10 CHIKV mono-infections and 2 CHIKV\/DENV co-infections, and distinguished four DENV serotypes in simulated matrices.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>A rapid and sensitive integrated method has been developed that combines duplex RT-MIRA for detecting DENV and CHIKV, and nested RT-MIRA for serotyping DENV. The simplicity and speed of the amplification and detection steps demonstrate this platform's potential for use in point-of-care testing and surveillance in areas with limited resources, particularly when used alongside portable extraction methods.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40249-026-01450-9","type":"journal-article","created":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T23:01:49Z","timestamp":1778281309000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A rapid multiplex platform for simultaneous detection of chikungunya virus, dengue virus, and dengue serotyping based on isothermal amplification and lateral flow dipsticks"],"prefix":"10.1186","volume":"15","author":[{"given":"Gaowen","family":"Liu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinlin","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yingchao","family":"Chang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mengyuan","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xueshan","family":"Xia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,5,9]]},"reference":[{"key":"1450_CR1","unstructured":"World Health Organization. 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