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Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Waterborne diseases represent a significant public health risk worldwide and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a genetic marker of the human-associated Bacteroides dorei for identification of human fecal pollution in ambient water samples. The following protocol includes water sample collection, filtration, DNA isolation with a sample processing control, qPCR amplification with an internal amplification control, and quality control data analysis.

A quantitative polymerase chain reaction (qPCR) method for the detection of enterococci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of water quality at recreational beaches. In this study we further examined the applicability of the method for analyses of diverse inland waters as well as tropical marine waters from Puerto Rico based on the frequencies of samples showing presumptive PCR interference. Interference was assessed by salmon DNA sample processing control (SPC) and internal amplification control (IAC) assay analysis results and pre-established acceptance criteria of <3.0 and <1.5 cycle threshold (Ct) offsets from control samples, respectively. SPC assay results were accepted in analyses of 93% of the inland water samples whereas the criterion was met at frequencies of 60% and 97% in analyses of samples from Puerto Rico in two different years of sampling. The functionality of the control assays and their acceptance criteria was assessed on the basis of relative recovery estimates of spiked enterococci target organisms extracted in the presence of water sample filters and sample-free control filters and was supported by observations that recovery estimates from the water sample and control filters were substantially different for samples that failed these criteria. Through the combined use of the SPC and IAC assays, two presumptive types of interference were identified. One type, observed in the tropical marine water samples, appeared to primarily affect the availability of the DNA templates for detection. The second type, observed in river water samples, appeared to primarily affect PCR amplification efficiency. In the presence of DNA template interference, adjustments from SPC assay results by the ΔΔCt comparative Ct calculation method decreased the variability of spiked enterococci recovery estimates and increased the similarity with control filters as compared to unadjusted recovery estimates obtained by the ΔCt calculation method. Use of a higher salmon DNA concentration in the extraction buffer also reduced this type of interference. The effects of amplification interference were largely reversed by dilution of the DNA extracts and even more effectively by the use of an alternative, commercial PCR reagent, designed for the analysis of environmental samples., (Published by Elsevier Ltd.)

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Background: Beach sand can harbor fecal indicator organisms and pathogens, but enteric illness risk associated with sand contact remains unclear., Methods: In 2007, visitors at 2 recreational marine beaches were asked on the day of their visit about sand contact. Ten to 12 days later, participants answered questions about health symptoms since the visit. F+ coliphage, Enterococcus, Bacteroidales, fecal Bacteroides, and Clostridium spp. in wet sand were measured using culture and molecular methods., Results: We analyzed 144 wet sand samples and completed 4999 interviews. Adjusted odds ratios (aORs) were computed, comparing those in the highest tertile of fecal indicator exposure with those who reported no sand contact. Among those digging in sand compared with those not digging in sand, a molecular measure of Enterococcus spp. (calibrator cell equivalents/g) in sand was positively associated with gastrointestinal (GI) illness (aOR = 2.0 [95% confidence interval (CI) = 1.2-3.2]) and diarrhea (2.4 [1.4-4.2]). Among those buried in sand, point estimates were greater for GI illness (3.3 [1.3-7.9]) and diarrhea (4.9 [1.8-13]). Positive associations were also observed for culture-based Enterococcus (colony-forming units/g) with GI illness (aOR digging = 1.7 [1.1-2.7]) and diarrhea (2.1 [1.3-3.4]). Associations were not found among nonswimmers with sand exposure., Conclusions: We observed a positive relationship between sand-contact activities and enteric illness as a function of concentrations of fecal microbial pollution in beach sand.

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3)copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R(2)≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters., (Published by Elsevier GmbH.)

Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed 'pooled' and 'mixed' for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the 'pooled' model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the 'mixed' model achieved uncertainty estimates similar to the 'single' model while increasing the number of available reaction wells per instrument run., (Published by Elsevier Ltd.)

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3 h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1-2499 CFU by MF culture analysis in Lake Michigan (N=56) and from 8 to 8695 cells by QPCR and 3-1941 CFU by MF culture in Lake Erie (N=47). Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68.

Monitoring for pathogenic Aspergillus species using a rapid, highly sensitive, quantitative polymerase chain reaction technique during carpet removal in a burn unit provided data that allowed patients to be safely returned to the refloored area sooner than if only conventional culture monitoring had been used.

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.