Invasive species monitoring based on eDNA multiplex PCR sequencing

Invasive species pose a significant threat to global biodiversity and the stability of ecosystems. Although environmental DNA (eDNA)-based quantitative PCR is considered effective, its limited multiplexing capacity makes it impractical for large-scale monitoring of invasive species. To address this limitation, we developed a novel and efficient approach for invasive species monitoring by combining multiplex PCR amplification with high-throughput sequencing. In this study, we screened 46 aquatic invasive species of major concern in China. We have integrated and designed 91 pairs of primers that can simultaneously amplify these species in a single PCR system. The validated method was applied to field monitoring in the Pearl River Basin to evaluate its practical performance. Multiplex PCR sequencing successfully detected 28 invasive species, with over 90% of environmental samples testing positive for invasive species DNA, demonstrating the method’s high sensitivity and broad applicability. Furthermore, all 11 invasive species identified through metabarcoding were also consistently detected by multiplex PCR sequencing, showing a strong positive correlation and high concordance across all monitoring sites. In conclusion, multiplex PCR sequencing represents a powerful and cost-effective tool for simultaneously detecting multiple aquatic invasive species in the early stages of invasion. It significantly improves detection efficiency, reduces monitoring costs and provides a solid foundation for developing a scientific and scalable monitoring system for aquatic invasive species.