FishChip: Development of a High-Throughput qPCR Assay for Freshwater Fish eDNA Quantification and Its Comparison With...

Quantitative monitoring of fish using environmental DNA (eDNA) typically relies on quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR), yet both methods are constrained in throughput, making it difficult to meet the demands of basin-scale surveys. High-throughput qPCR (HT-qPCR) has emerged as a platform capable of simultaneously processing large numbers of targets and samples, but its performance for fish eDNA quantification and its comparability with ddPCR remain poorly characterized. Here, we selected nine widely distributed freshwater fishes and designed 19 species-specific primer pairs (2-3 per species). All assays performed well on a high-throughput chip-based qPCR (HT-qPCR) platform, with amplification efficiencies ranging from 91.07% to 105.83% and 78.9% of assays falling within the optimal 95%-105% interval. Applying HT-qPCR to quantify fish eDNA across six aquatic regions, Cyprinus carpio (C. carpio) achieved a 100% detection rate. For the same fish species, the two primer sets exhibited measurable differences in detection rate and quantitative estimates. HT-qPCR and ddPCR estimates were strongly correlated (r = 0.858), and Deming regression indicated near-equivalence (HT-qPCR = 0.52 + 0.99 × ddPCR). Bland-Altman analysis revealed a consistent ~3-fold overestimation by HT-qPCR relative to ddPCR, which is generally considered the more accurate method, suggesting that a correction factor could harmonize data across platforms. These results validate the utility of HT-qPCR for catchment-scale quantification of fish eDNA concentrations and provide methodological innovation and technical support for aquatic ecosystem monitoring and assessment.