Integrating RPA-LFD and TaqMan qPCR for Rapid On-Site Screening and Accurate Laboratory Identification of Coilia brachygnathus and Coilia nasus 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.