Domestic wastewater influent profiling using mitochondrial real-time PCR for source tracking animal contamination

Real-time PCR amplifying mammalian and avian mitochondrial DNA (mtDNA) was developed to characterize domestic and light industrial wastewater influent from two municipal wastewater treatment facilities (WWTF) over a 24-week period. Influent samples were assayed with species-specific primers and dual-labeled probes for human, bovine, swine, dog, cat, Canada goose and white-tailed deer to detect and quantify eukaryotic mtDNA contributors to local municipal wastewaters. Human (mean = 9.6 × 104 copies/ml) and dog (mean = 5.3 × 102 copies/ml) mtDNA were detected in all 24 samples (12 samples/site). Bovine and swine mtDNA were detected sporadically and at lower levels than human mtDNA, means = 3.0 × 104 and 9.5 × 102 copies/ml, respectively. Domestic cat, Canada goose and white-tailed deer were detected only once in 24 samples. Mitochondrial DNA concentrations were compared to other bacterial, chemical and spectrophotometric parameters. Human mtDNA was positively correlated with ammonia concentration (P = 0.01) and initial OD600 reading (P = 0.02) at one WWTF. Bovine mtDNA was positively correlated with biological oxygen demand (BOD) (P = 0.02), final DNA concentration (P = 0.03), initial and final humic acid concentrations (P = 0.01, P = 0.01), and final OD600 (P = 0.03) at one WWTF and total suspended solids (TSS) (P = 0.04, P = 0.09) at both facilities. Fecal coliforms were not positively or negatively correlated with mtDNA concentrations of any species assayed. For source tracking purposes, a combination of human (105 copies/ml) and dog mtDNA signal (102 copies/ml) could be indicative of municipal domestic wastewater contamination of environmental waters.