Soil eDNA-based droplet digital PCR assay as a new monitoring tool for terrestrial mammals

This study demonstrates the effectiveness of droplet digital PCR (ddPCR) for identifying vertebrate taxa from soil-derived environmental DNA (eDNA) a methodological combination not previously documented in literature. The protocol is optimized for detecting cryptic species of the lesser blind mole rat (BMR). A total of 38 soil samples were collected from five cryptic BMR species across 12 localities in Serbia. Species-specific primer/probe sets were designed based on the cytochrome b (cytb) gene. After optimization of ddPCR assays and verification of primer/probe specificity, all samples were amplified in triplicates and the results’ significance was tested using the RStudioV4.3.3 Environment. The total number of positive findings was detected in 33 out of 38 samples (86.84 %), highlighting the strength of ddPCR in detecting and quantifying rare and degraded target DNA from soil samples. Concentration values varied from 0.073 to 236 copies/µL. The primer/probe sets were highly specific and the only difficulty emerged in distinguishing between two phylogenetically closest cryptic species, but with a discrepancy in fluorescence amplitudes in specific (target) and non-specific (non-target) cryptic species. We have confirmed that soil eDNA-based ddPCR species identification is a fast and highly sensitive non-invasive monitoring method. It allows quick and easy sampling of many locations across wide areas, providing essential data for conservation management. This protocol offers a valuable framework for conservation strategies targeting cryptic species, while also serving as a foundation for adaptation and broader implementation across other terrestrial vertebrates of concern – particularly those that are strictly protected or classified as endangered.––