Improved real-time PCR assays for the detection of fecal indicator bacteria in surface waters with different instrument and reagent systems

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (notemplate and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark. Fecal pollution remains a significant challenge for ambient water quality managers worldwide. In the United States alone, it is estimated that 39.2% of all rivers, lakes, and streams are unsafe for recreational use, with fecal pathogens as the number one cause of impairment (1). General fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, shed by nearly all warm-blooded animals, are routinely used to assess water quality. However, general FIB cannot discriminate between different animal groups, making it challenging to pinpoint the origin of fecal pollution. Researchers and managers alike recognize the advantages of animal source information to help solve long-standing ambient water quality problems. As a result, a number of fecal source identification technologies have been developed (2-9). These methods have been employed to address challenges such as the identification ...

Section: Discussionmentioning

confidence: 99%

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

Shanks

Kelty

Oshiro

et al. 2016

“…All amplifications were performed in a 7900 HT Fast real-time sequence detector (Applied Biosystems). Reaction conditions and thermal cycling parameters for CowM2, CowM3, GenBac3, EC23S857, and Entero1 are reported elsewhere (14,17,18). For CampF2 Experiment controls.…”

Section: Methodsmentioning

confidence: 99%

Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces

Shanks

Kelty

Peed

et al. 2014

cCalves 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.

“…The TaqMan probe sequence was 5=-6-carboxyfluorescein (FAM)-CAATATTCCTCACTGCTGCCTCCCGTA-BHQ-1-3= (37). All reactions were carried out on a model 7300 Real Time PCR system (Life Technologies, Grand Island, NY), using previously published thermocycler conditions (37). A plasmid containing the target gene sequence (IDT-DNA, Coralville, IA) was used as a positive control in all reaction mixtures.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Weidhaas

Mantha

Hair

et al. 2015

bWater quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml ؊1 and 2 log gene copies g litter ؊1 under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.