Integrating RPA-LFD and TaqMan qPCR for Rapid On-Site Screening and Accurate Laboratory Identification of <em>Coilia brachygnathus</em> and <em>Coilia nasus</em> in the Yangtze River

Accurately differentiating between Coilia brachygnathus and Coilia nasus is critical for fisheries management, conservation efforts, and combating market fraud. Current morphological identifi-cation remains challenging due to their high morphological similarity—particularly for processed samples—while conventional molecular methods often lack the speed or specificity required for field applications or high-throughput screening. Here, we developed and validated a novel inte-grated approach that combines TaqMan quantitative real-time PCR (qPCR) for precise genotyping of C. brachygnathus and C. nasus with Recombinase Polymerase Amplification coupled with Lateral Flow Dipstick (RPA-LFD) for rapid on-site screening. First, species-specific RPA-LFD assays were designed to target the mitochondrial COI gene sequence. This enabled visual detection within 10 minutes at 37°C, with a sensitivity of 10² copies/μL, and required no complex equipment. A dual TaqMan MGB qPCR assay was further developed by validating stable differentiating SNPs (chr21:3798155, C/T) between C. brachygnathus and C. nasus, using FAM/VIC dual-labeled MGB probes. Results showed that this assay could distinguish the two species in a single tube: for C. brachygnathus, Ct values in the FAM channel were significantly earlier than those in the VIC channel (ΔCt≥1), with a FAM detection limit of 125 copies/reaction; for C. nasus, only VIC channel ampli-fication was observed, with a detection limit as low as 12.5 copies/reaction. Validation with 171 known tissue samples demonstrated 100% concordance with expected species identities. This in-tegrated approach effectively combines the high accuracy and quantitative capacity of TaqMan qPCR for confirmatory laboratory genotyping with the speed, simplicity, and portability of RPA-LFD for initial field or point-of-need screening. This reliable, efficient, and user-friendly technique provides a powerful tool for resource management, biodiversity monitoring, and en-suring the authenticity of high-quality C. brachygnathus and C. nasus.