Development of new eDNA assays for the detection of two European fire-bellied toads (Bombina) via single-species and parallel High-Throughput real time PCR

We developed and validated two species-specific eDNA assays for the locally endangered yellow-bellied toad ( Bombina variegata ) and the red-bellied toad ( B. bombina ) with comprehensive in silico, in vitro, and in vivo testing. We evaluated assay performance on field samples using single-species qPCR and High-Throughput qPCR (HT-qPCR) on a Biomark HD system with different master mix chemistries. Analytical workflow significantly affected Ct values with HT-qPCR yielding consistently lower Ct values than qPCR due to prior preamplification. Both HT-qPCR chemistries showed comparable detection performance with consistent and reproducible results across field samples. For B. variegata -samples HT-qPCR workflows substantially improved detection rates. The new assays are suitable for applied eDNA monitoring and can serve as robust complement to conventional amphibian monitoring programs.