Low‐Concentration Enhancement Effect: Enabling Sensitive Detection of EML4 ‐ ALK Variants via Multiplex RT ‐ MIRA

{"OBJECTIVE:":[0],"ALK":[1],"fusion":[2],"variant":[3,35,137],"detection":[4,45,67,178,198],"is":[5],"essential":[6],"for":[7,43,66,135,187,205],"guiding":[8],"targeted":[9],"therapy":[10],"in":[11,30],"non-small":[12],"cell":[13],"lung":[14],"cancer":[15],"(NSCLC).":[16],"The":[17,127],"emerging":[18],"reverse":[19],"transcription":[20],"multi-enzyme":[21],"isothermal":[22],"rapid":[23,194],"amplification":[24],"(RT-MIRA)":[25],"technology":[26],"exhibits":[27],"substantial":[28],"potential":[29],"molecular":[31],"diagnostics,":[32],"enabling":[33],"efficient":[34],"profiling.":[36],"However,":[37],"its":[38],"performance":[39],"and":[40,55,61,74,120,195,200],"optimization":[41,185],"strategies":[42],"multiplex":[44,64,100,129,188],"remain":[46],"inadequately":[47],"characterized.":[48],"This":[49],"study":[50,87,191],"therefore":[51],"aimed":[52],"to":[53],"analyze":[54],"optimize":[56],"the":[57,83,91,96,99,105,108,151],"RT-MIRA":[58,101,130],"reaction":[59,109],"system":[60],"develop":[62],"a":[63,140,162,173,183,193,202],"assay":[65,131,152],"of":[68,98,107,209],"EML4-ALK":[69,197],"variants":[70],"(including":[71],"V1,":[72],"V2,":[73],"V3a/b":[75],"variants)":[76],"with":[77,156],"high":[78],"sensitivity.":[79,179],"METHODS:":[80],"Breaking":[81],"from":[82],"conventional":[84],"approaches,":[85],"this":[86,190],"prioritized":[88],"sensitivity":[89,134],"as":[90],"primary":[92],"evaluation":[93],"metric":[94],"during":[95],"development":[97],"assay,":[102],"while":[103],"ensuring":[104],"specificity":[106],"system.":[110],"Reaction":[111],"conditions":[112],"were":[113],"systematically":[114],"optimized":[115,128],"by":[116],"combining":[117],"time-to-threshold":[118],"(TT)":[119],"relative":[121],"fluorescence":[122],"intensity":[123],"(RFI)":[124],"analyses.":[125],"RESULTS:":[126],"achieved":[132],"single-copy":[133],"each":[136],"plasmid.":[138],"In":[139],"preliminary":[141],"validation":[142],"using":[143],"21":[144],"archived":[145],"formalin-fixed":[146],"paraffin-embedded":[147],"(FFPE)":[148],"clinical":[149,207],"samples,":[150],"demonstrated":[153],"complete":[154],"concordance":[155],"commercial":[157],"kits.":[158],"Notably,":[159],"we":[160],"observed":[161],"\\"low-concentration":[163],"enhancement":[164],"effect,\\"":[165],"where":[166],"reducing":[167],"primer":[168],"or":[169],"probe":[170],"concentrations":[171],"within":[172],"specific":[174],"range":[175],"markedly":[176],"improved":[177],"CONCLUSION:":[180],"By":[181],"developing":[182],"systematic":[184],"strategy":[186],"RT-MIRA,":[189],"provides":[192],"sensitive":[196],"tool":[199],"establishes":[201],"methodological":[203],"foundation":[204],"broader":[206],"applications":[208],"RT-MIRA.":[210]}

Authors

Institutions

Publication Details

Journal
Journal of Clinical Laboratory Analysis
Published
2026-09-20
DOI
https://doi.org/10.1002/jcla.70351
Primary Topic
Lung Cancer Treatments and Mutations
Type
article
Field-Weighted Citation Impact
0.00
Controls
|||
ALL TIME
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
article

Low‐Concentration Enhancement Effect: Enabling Sensitive Detection of EML4 ‐ ALK Variants via Multiplex RT ‐ MIRA

Luo Xi-yao, Qing Yu Huang, Xiaodong Ren, Xian-Ge Sun et al.
Journal of Clinical Laboratory Analysis
Lung Cancer Treatments and Mutations
article

Low‐Concentration Enhancement Effect: Enabling Sensitive Detection of EML4 ‐ ALK Variants via Multiplex RT ‐ MIRA

Luo Xi-yao, Qing Yu Huang, Xiaodong Ren, Xian-Ge Sun, Qiushi Wang, Chunyan Wang, Hualiang Xiao, Hao-Zheng Li, Jin-Mi Li, Jing Lv, Hong Wang
article en

Abstract

OBJECTIVE: ALK fusion variant detection is essential for guiding targeted therapy in non-small cell lung cancer (NSCLC). The emerging reverse transcription multi-enzyme isothermal rapid amplification (RT-MIRA) 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-MIRA reaction system and develop a multiplex assay for detection of EML4-ALK variants (including V1, V2, and V3a/b variants) with high sensitivity. METHODS: Breaking from the conventional approaches, this study prioritized sensitivity as the primary evaluation metric during the development of the multiplex RT-MIRA assay, while ensuring the specificity of the reaction system. Reaction conditions were systematically optimized by combining time-to-threshold (TT) and relative fluorescence intensity (RFI) analyses. RESULTS: The optimized multiplex RT-MIRA assay achieved single-copy sensitivity for each variant plasmid. In a preliminary validation using 21 archived formalin-fixed paraffin-embedded (FFPE) clinical samples, the assay demonstrated complete concordance with commercial kits. Notably, we observed a "low-concentration enhancement effect," where reducing primer or probe concentrations within a specific range markedly improved detection sensitivity. CONCLUSION: By developing a systematic optimization strategy for multiplex RT-MIRA, this study provides a rapid and sensitive EML4-ALK detection tool and establishes a methodological foundation for broader clinical applications of RT-MIRA.

Journal of Clinical Laboratory Analysis
Army Medical University (CN), Academy of Military Medical Sciences (CN), Daping Hospital (CN)
Openalex Percentile: Top 11%
Lung Cancer Treatments and Mutations
AI Navigator

Ask Laika to Summarize, Analyze, and Connect papers live on the map.

Summarize Papers & Methodologies

Extract key findings, datasets, and comparative methods across publications.

Benchmark Rankings & Visual Analytics

Rank top research institutions, authors, funders, topics, and journals by Field-Weighted Citation Impact (FWCI) and paper volume with instant charts.

Connect Distant Disciplines

Bridge topological clusters on the map to find hidden collaborative intersections.