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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. [ABSTRACT FROM AUTHOR]

Two bacteriophages, ϕ6 and ϕ8, were investigated as potential surrogates for H5N1 highly pathogenic avian influenza virus in persistence and chlorine inactivation studies in water. In the persistence studies, ϕ6 and ϕ8 remained infectious at least as long as the H5N1 viruses at both 17 and 28°C in fresh water, but results varied in salinated water. The bacteriophage ϕ6 also exhibited a slightly higher chlorine resistance than that of the H5N1 viruses. Based upon these findings, the bacteriophages may have potential for use as surrogates in persistence and inactivation studies in fresh water. [ABSTRACT FROM AUTHOR]

The objective of this study was to measure hydroxyl radical reaction rates using both UV/H[sub2]O[sub2] and pulse radiolysis techniques for 10 US EPA Contaminant Candidate List compounds (2,6- and 2,4-DNT, EPTC, prometon, linuron, diuron, RDX, molinate, nitrobenzene, and terbacil). The rate constants determined using these techniques were compared to each other and to values reported in the literature. Difference factors between k[subOH] obtained using UV/H[sub2]O[sub2] and pulse radiolysis ranged from 1.1 to 4.7. It was shown that even small differences in hydroxyl radical rate constants values can result in fairly large differences (up to 50%) when trying to predict removals of pollutants in an advanced oxidation process. [ABSTRACT FROM AUTHOR]

Because lot variability is accounted for in the current Long Term 2 Enhanced Surface Water Treatment Rule, water utilities may want to use a similar safety factor for a sequential inactivation process to inactivate C. parvum oocysts with ozone. [ABSTRACT FROM AUTHOR]

Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. in these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achieve a high degree of inactivation. Different lots of Cryptosporidium parvum oocysts are used in these studies, and the inactivation rate may vary depending on the lot of oocysts used. The goal of this paper is to account for the error in estimating the amount of inactivation after exposure to UV radiation, and for the effect of lot variability in determining the required UV radiation. A Bayesian approach is used to simultaneously model the logistic dose-response model and the UV inactivation kinetic model. The oocysts lot variability is incorporated using a hierarchical Bayesian model. Posterior distributions using Markov Chain Monte Carlo method is used to obtain estimates and Bayesian credible interval for the required UV radiation to achieve a given inactivation level of Cryptosporidium parvum oocysts. [ABSTRACT FROM AUTHOR]

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods. [ABSTRACT FROM AUTHOR]

Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values., (Published by Elsevier B.V.)

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.