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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.

To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5[degrees]C. [...]

Abstract Background In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. Results Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. Conclusion The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.

A performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays which are associated with either ruminant or bovine feces are reported. The large discrepancies in the performance of bovine-associated assays across different bovine populations and variability in assay performance between host populations indicated that the use of bovine microbial source-tracking applications would need a prior characterization at each water-shed of interest.

The quantitative PCR arrays are developed for the quantification of two human-specific genetic markers targeting Bacteriodales-like cell surface-associated genes. Assay performance combined with the prevalence of DNA targets in sewage samples have provided evidence that has supported the application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

Quantitative PCR assays are developed for the detection of bovine feces-specific genetic markers and a method is designed for the enumeration of these markers by using a Markov chain Monte Carlo approach. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations have shown that these quantitative assays are useful in monitoring bovine fecal pollution in ambient waters.

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]

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]