Identifying sources of fecal contamination is crucial for effective management of water resources, as it enables targeted remediation strategies and therefore helps prevent the spread of disease. The origin of fecal contamination can be identified by detecting host-associated molecular markers derived from gut microbiota. In this study, we focused on developing new assays for detecting animal-derived sources of fecal pollution. To achieve this, we collected more than 700 samples of animal feces and fecal waste from livestock (manure, slurry, and liquid manure) from over 50 animal species across Slovenia. Total DNA was extracted and the V3–V4 hypervariable region of the 16S rRNA gene was sequenced to characterize microbial communities. We developed MicrobiomePrime, an automated bioinformatic tool for designing primer pairs targeting host-associated markers with high sensitivity and specificity. Using MicrobiomePrime, we designed PCR assays for detecting fecal contamination from cattle, pigs, dogs, and nutria, as well as assays targeting fecal waste from cattle and pigs. Six of the nine selected assays achieved 100% sensitivity when tested on five to ten target samples, while eight showed specificity ≥ 90%, indicating high reliability. Validation on environmental water samples demonstrated that these assays complement existing methods for fecal pollution diagnostics by enabling detection of a broader range of host sources without producing false-positive results in uncontaminated environments.
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