Dataset Information

Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution.

ABSTRACT: Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10(6) copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.

SUBMITTER: Shanks OC

PROVIDER: S-EPMC2227726 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution.

Shanks Orin C OC Atikovic Emina E Blackwood A Denene AD Lu Jingrang J Noble Rachel T RT Domingo Jorge Santo JS Seifring Shawn S Sivaganesan Mano M Haugland Richard A RA

Applied and environmental microbiology 20071207 3

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10(6) copies of target DNA, with a coeffi ...[more]

PMID: 18065617

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Project description:The human microbiome contains many organisms that could potentially be used as indicators of human fecal pollution. Here we report the development of two novel human-associated genetic marker assays that target organisms within the family Lachnospiraceae Next-generation sequencing of the V6 region of the 16S rRNA gene from sewage and animal stool samples identified 40 human-associated marker candidates with a robust signal in sewage and low or no occurrence in samples from nonhuman hosts. Two were chosen for quantitative PCR (qPCR) assay development using longer sequences (the V2 to V9 regions) generated from clone libraries. Validation of these assays with these markers, designated Lachno3 and Lachno12, was performed using fecal samples (n = 55) from cat, dog, pig, cow, deer, and gull sources, and the results were compared with those of established host-associated assays (the Lachno2 marker and two human Bacteroides markers, the HB and HF183/BacR287). Each of the established assays cross-reacted with samples from at least one other animal species, including animals common in urban areas. The Lachno3 and Lachno12 markers were primarily human associated; however, the Lachno12 marker demonstrated low levels of cross-reactivity with samples from select cows and nonspecific amplification with samples from pigs. This limitation may not be problematic when testing urban waters. These novel markers resolved ambiguous results from previous investigations of stormwater-impacted waters, demonstrating their utility. The complexity of the microbiome in humans and animals suggests that no single organism is strictly specific to humans, and the use of multiple complementary markers in combination will provide the highest resolution and specificity for assessing fecal pollution sources.IMPORTANCE Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.

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 for detection and enumeration of genetic markers of bovine fecal pollution.

Publications

Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution.

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