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Published on: December 3, 2011
Domestic wastewater influent profiling using mitochondrial real-time PCR for source tracking animal contamination
Jane M Caldwell1, Jay F Levine
1Department of Population Health and Pathobiology, Aquatic Epidemiology and Conservation Laboratory, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, United States.
Abstract:
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 x 10(4) copies/ml) and dog (mean=5.3 x 10(2) 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 x 10(4) and 9.5 x 10(2) 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 OD(600) 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 OD(600) (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 (10(5) copies/ml) and dog mtDNA signal (10(2) copies/ml) could be indicative of municipal domestic wastewater contamination of environmental waters.
Insights
A new real-time PCR method detected human and dog mitochondrial DNA (mtDNA) in wastewater, indicating domestic contamination. This method can help track wastewater sources in environmental waters.
Area of Science:
- Environmental Science
- Molecular Biology
- Analytical Chemistry
Background:
- Wastewater characterization is crucial for environmental monitoring.
- Identifying sources of contamination in municipal wastewater is challenging.
- Mitochondrial DNA (mtDNA) offers a potential marker for species-specific source tracking.
Purpose of the Study:
- To develop and validate a real-time PCR assay for detecting and quantifying mammalian and avian mitochondrial DNA (mtDNA) in wastewater influent.
- To characterize the eukaryotic mtDNA contributors in wastewater from two municipal wastewater treatment facilities (WWTF) over a 24-week period.
- To correlate mtDNA concentrations with other wastewater parameters for source tracking applications.
Main Methods:
- Developed real-time PCR assays using species-specific primers and dual-labeled probes.
- Assayed influent samples for human, bovine, swine, dog, cat, Canada goose, and white-tailed deer mtDNA.
- Collected samples from two WWTFs over 24 weeks and analyzed alongside bacterial, chemical, and spectrophotometric data.
Main Results:
- Human mtDNA (mean=9.6 x 10^4 copies/ml) and dog mtDNA (mean=5.3 x 10^2 copies/ml) were consistently detected in all samples.
- Bovine and swine mtDNA were found sporadically at lower concentrations.
- Human mtDNA correlated positively with ammonia and initial OD(600); bovine mtDNA correlated with BOD, humic acid, OD(600), and TSS.
Conclusions:
- Real-time PCR is effective for detecting and quantifying diverse eukaryotic mtDNA in wastewater.
- A combination of human and dog mtDNA signals can indicate domestic wastewater contamination.
- This method provides a valuable tool for source tracking in environmental water assessments.
