Dataset Information

Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions.

ABSTRACT: A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 x 10(9) marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10(5) marker equivalents per liter).

SUBMITTER: Reischer GH

PROVIDER: S-EPMC1538736 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions.

Reischer Georg H GH Kasper David C DC Steinborn Ralf R Mach Robert L RL Farnleitner Andreas H AH

Applied and environmental microbiology 20060801 8

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 x 10(9) marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples ...[more]

PMID: 16885315

Similar Datasets

Project description:Alternative indicators have been developed that can be used to identify host sources of fecal pollution, yet little is known about how their distribution and fate compare to traditional indicators. Escherichia coli and enterococci were widely distributed at the six beaches studied and were detected in almost 95% of water samples (n = 422) and 100% of sand samples (n = 400). Berm sand contained the largest amount of E. coli (P < 0.01), whereas levels of enterococci were highest in the backshore (P < 0.01). E. coli and enterococci were the lowest in water, using a weight-to-volume comparison. The gull-associated Catellicoccus marimammalium (Gull2) marker was found in over 80% of water samples, regardless of E. coli levels, and in 25% of sand samples. Human-associated Bacteroides (HB) and Lachnospiraceae (Lachno2) were detected in only 2.4% of water samples collected under baseflow and post-rain conditions but produced a robust signal after a combined sewage overflow, despite low E. coli concentrations. Burdens of E. coli and enterococci in water and sand were disproportionately high in relation to alternative indicators when comparing environmental samples to source material. In microcosm studies, Gull2, HB, and Lachno2 quantitative PCR (qPCR) signals were reduced twice as quickly as those from E. coli and enterococci and approximately 20% faster than signals from culturable E. coli High concentrations of alternative indicators in source material illustrated their high sensitivity for the identification of fecal sources; however, differential survival and the potential for long-term persistence of traditional fecal indicators complicate the use of alternative indicator data to account for the levels of E. coli and enterococci in environmental samples.E. coli and enterococci are general indicators of fecal pollution and may persist in beach sand, making their use problematic for many applications. This study demonstrates that gull fecal pollution is widespread at Great Lakes beaches, whereas human and ruminant contamination is evident only after major rain events. An exploration of sand as a reservoir for indicators found that E. coli was ubiquitous, while gull host markers were detected in only 25% of samples. In situ sand beach microcosms provided decay rate constants for E. coli and enterococci relative to alternative indicators, which establish comparative benchmarks that would be helpful to distinguish recent from past pollution. Overall, alternative indicators are useful for identifying sources and assessing potentially high health risk contamination events; however, beach managers should be cautious in attempting to directly link their detection to the levels of E. coli or enterococci.

Project description:Microbial contamination of recreation waters is a major concern globally, with pollutants originating from many sources, including human and other animal wastes often introduced during storm events. Fecal contamination is traditionally monitored by employing culture methods targeting fecal indicator bacteria (FIB), namely E. coli and enterococci, which provides only limited information of a few microbial taxa and no information on their sources. Host-associated qPCR and metagenomic DNA sequencing are complementary methods for FIB monitoring that can provide enhanced understanding of microbial communities and sources of fecal pollution. Whole metagenome sequencing (WMS), quantitative real-time PCR (qPCR), and culture-based FIB tests were performed in an urban watershed before and after a rainfall event to determine the feasibility and application of employing a multi-assay approach for examining microbial content of ambient source waters. Cultivated E. coli and enterococci enumeration confirmed presence of fecal contamination in all samples exceeding local single sample recreational water quality thresholds (E. coli, 410 MPN/100 mL; enterococci, 107 MPN/100 mL) following a rainfall. Test results obtained with qPCR showed concentrations of E. coli, enterococci, and human-associated genetic markers increased after rainfall by 1.52-, 1.26-, and 1.11-fold log10 copies per 100 mL, respectively. Taxonomic analysis of the surface water microbiome and detection of antibiotic resistance genes, general FIB, and human-associated microorganisms were also employed. Results showed that fecal contamination from multiple sources (human, avian, dog, and ruminant), as well as FIB, enteric microorganisms, and antibiotic resistance genes increased demonstrably after a storm event. In summary, the addition of qPCR and WMS to traditional surrogate techniques may provide enhanced characterization and improved understanding of microbial pollution sources in ambient waters.

Dataset Information

Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions.

Publications

Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets