qPCR validation and field application of a rapid RPA-LFD assay for monitoring the invasive alligator gar (Atractosteus spatula)

Understanding species’ distributions beyond their native ranges is essential for monitoring non-native species occurrences. The alligator gar ( Atractosteus spatula ), native to North America and listed as a key invasive species in China’s Catalogue of Key Managed Invasive Alien Species, poses significant threats to aquatic ecosystems. To enable rapid on-site detection, we developed and validated novel molecular assays based on recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and quantitative PCR (qPCR). We designed species-specific primers and probes, optimized reaction conditions, and evaluated assay performance. Both RPA-LFD and qPCR exhibited high sensitivity, with detection limits as low as 4 copies/μL, and strong specificity for A. spatula without cross-reactivity to non-target species. Under optimized conditions, the RPA-LFD assay achieved results within 15 minutes at 38 °C. Field testing in a natural breeding pond confirmed reliable detection, and the best-performing RPA assay (RPAS1) showed comparable efficiency to the qPCR gold standard. This study provides robust, field-deployable tools that empower early detection and proactive monitoring of invasive species, with strong potential for application in citizen science and large-scale biodiversity surveillance. Graphic abstract