Fecha de publicación: -- Fuente: PubMed "industrial biotechnology" Anal Chim Acta. 2026 Dec 15;1427:346423. doi: 10.1016/j.aca.2026.346423. Epub 2026 Oct 21.ABSTRACTMultiplex nucleic-acid analysis requires orthogonal signal channels capable of resolving multiple targets without substantial cross-interference. Here, an RPA-assisted three-channel CRISPR fluorescence readout was developed by coupling the distinct collateral-cleavage activities of LwaCas13a, PsmCas13b, and LbaCas12a to FAM-, ROX-, and HEX-based reporters, respectively, for detection of Venezuelan equine encephalitis virus (VEEV), tick-borne encephalitis virus (TBEV), and an eastern/western equine encephalitis virus (EEEV/WEEV) group target. The corresponding singleplex assays showed no detectable cross-reactivity with the tested non-target viral templates and achieved operational analytical detection limits of 10 copies/μL for VEEV and TBEV and 100 copies/μL for the EEEV/WEEV group assay. Integration of the three Cas-crRNA-reporter modules into one downstream reaction enabled discrimination of single-, dual-, and three-target combinations, with an analytical detection limit of 100 copies/μL for each target in the equal-concentration three-target mixture. Reporter optimization provided balanced channel responses, while inter-day repeatability and interlaboratory reproducibility yielded coefficients of variation below 10%. In a preliminary matrix evaluation using contrived equine nasal-swab samples, plasmid-spiked samples and unspiked negative controls showed concordant classification between the CRISPR readout and RT-qPCR, indicating compatibility of the assay workflow with the tested sample matrix. The method provides a three-channel analytical architecture that consolidates three downstream CRISPR readouts into one reaction while retaining target-assigned fluorescence interpretation.PMID:43003575 | DOI:10.1016/j.aca.2026.346423