{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T11:41:26Z","timestamp":1792755686337,"version":"4.5.0"},"reference-count":53,"publisher":"Wiley","issue":"20","license":[{"start":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T00:00:00Z","timestamp":1789862400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T00:00:00Z","timestamp":1789862400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82172370"],"award-info":[{"award-number":["82172370"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Clinical Laboratory Analysis"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>ALK fusion variant detection is essential for guiding targeted therapy in non\u2010small cell lung cancer (NSCLC). The emerging reverse transcription multi\u2010enzyme isothermal rapid amplification (RT\u2010MIRA) technology exhibits substantial potential in molecular diagnostics, enabling efficient variant profiling. However, its performance and optimization strategies for multiplex detection remain inadequately characterized. This study therefore aimed to analyze and optimize the RT\u2010MIRA reaction system and develop a multiplex assay for detection of EML4\u2010ALK variants (including V1, V2, and V3a\/b variants) with high sensitivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Breaking from the conventional approaches, this study prioritized sensitivity as the primary evaluation metric during the development of the multiplex RT\u2010MIRA assay, while ensuring the specificity of the reaction system. Reaction conditions were systematically optimized by combining time\u2010to\u2010threshold (TT) and relative fluorescence intensity (RFI) analyses.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimized multiplex RT\u2010MIRA assay achieved single\u2010copy sensitivity for each variant plasmid. In a preliminary validation using 21 archived formalin\u2010fixed paraffin\u2010embedded (FFPE) clinical samples, the assay demonstrated complete concordance with commercial kits. Notably, we observed a \u201clow\u2010concentration enhancement effect,\u201d where reducing primer or probe concentrations within a specific range markedly improved detection sensitivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>By developing a systematic optimization strategy for multiplex RT\u2010MIRA, this study provides a rapid and sensitive EML4\u2010ALK detection tool and establishes a methodological foundation for broader clinical applications of RT\u2010MIRA.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/jcla.70351","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T06:16:18Z","timestamp":1789971378000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Low\u2010Concentration Enhancement Effect: Enabling Sensitive Detection of\n                    <scp>EML4<\/scp>\n                    \u2010\n                    <scp>ALK<\/scp>\n                    Variants via Multiplex\n                    <scp>RT<\/scp>\n                    \u2010\n                    <scp>MIRA<\/scp>"],"prefix":"10.1002","volume":"40","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1172-5579","authenticated-orcid":false,"given":"Xiao\u2010Dong","family":"Ren","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine Daping Hospital, Army Medical University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi\u2010Yao","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine Daping Hospital, Army Medical University  Chongqing China"},{"name":"Academy of Military Medical Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin\u2010Mi","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine 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