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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]

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]

Periphyton is a complex mixture of algae, microbes, inorganic sediment, and organic matter that is attached to submerged surfaces in most flowing freshwater systems. This natural community is known to absorb pollutants from the water column, resulting in improved water quality. However, the role of periphyton in the fate and transport of genetic fecal markers suspended in the water column remains unclear. As application of genetic-based methodologies continues to increase in freshwater settings, it is important to identify any interactions that could potentially confound water quality interpretations. A 16 week indoor mesocosm study was conducted to simultaneously measure genetic fecal markers in the water column and in the associated periphyton when subject to wastewater source loading. Treated wastewater effluent was pumped directly from a treatment facility adjacent to the experimental stream facility. Inflow and outflow surface water grabs were paired with the collection of periphyton samples taken from the mesocosm substrates on a weekly basis. Samples were analyzed with three genetic fecal indicator quantitative real-time polymerase chain reaction assays targeting Escherichia coli (EC23S857), enterococci (Entero1), and Bacteroidales (GenBac3), as well as, two human host-associated fecal pollution markers (HF183 and HumM2). In addition, periphyton dry mass was measured. During wastewater effluent loading, genetic markers were detected in periphyton at frequencies up to 100% (EC23S857, Entero1, and GenBac3), 59.4% (HF183), and 21.9% (HumM2) confirming sequestration from the water column. Mean net-flux shifts in water column inflow and outflow genetic indicator concentrations further supported interactions between the periphyton and water column. In addition, positive correlations were observed between periphyton dry mass and genetic marker concentrations ranging from r = 0.693 (Entero1) to r = 0.911 (GenBac3). Overall, findings support the notion that genetic markers suspended in the water column can be trapped by periphyton, further suggesting that the benthic environment in flowing freshwater systems may be an important factor to consider for water quality management with molecular methods., Competing Interests: The authors declare no competing financial interest.