Kriston M. Strickler | Search USGS

USGS2012

Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

West Virginia↗

USGS2007

Presumptive Sources of Fecal Contamination in Four Tributaries to the New River Gorge National River, West Virginia, 2004

Several methods were used to determine the sources of fecal contamination in water samples collected during September and October 2004 from four tributaries to the New River Gorge National River -- Arbuckle Creek, Dunloup Creek, Keeney Creek, and Wolf Creek. All four tributaries historically have had elevated levels of fecal coliform bacteria. The source-tracking methods used yielded various results, possibly because one or more methods failed. Sourcing methods used in this study included the detection of several human-specific and animal-specific biological or molecular markers, and library-dependent pulsed-field gel electrophoresis analysis that attempted to associate Escherichia coli bacteria obtained from water samples with animal sources by matching DNA-fragment banding patterns. Evaluation of the results of quality-control analysis indicated that pulsed-field gel electrophoresis analysis was unable to identify known-source bacteria isolates. Increasing the size of the known-source library did not improve the results for quality-control samples. A number of emerging methods, using markers in Enterococcus, human urine, Bacteroidetes, and host mitochondrial DNA, demonstrated some potential in associating fecal contamination with human or animal sources in a limited analysis of quality-control samples. All four of the human-specific markers were detected in water samples from Keeney Creek, a watershed with no centralized municipal wastewater-treatment facilities, thus indicating human sources of fecal contamination. The human-specific Bacteroidetes and host mitochondrial DNA markers were detected in water samples from Dunloup Creek, Wolf Creek, and to a lesser degree Arbuckle Creek. Results of analysis for wastewater compounds indicate that the September 27 sample from Arbuckle Creek contained numerous human tracer compounds likely from sewage. Dog, horse, chicken, and pig host mitochondrial DNA were detected in some of the water samples with the exception of the October 5 sample from Dunloup Creek. Cow, white-tailed deer, and Canada goose DNA were not detected in any of the samples collected from the four tributaries, despite the presence of these animals in the watersheds. Future studies with more rigorous quality-control analyses are needed to investigate the potential applicability and use of these emerging methods. Because many of the detections for the various methods could vary over time and with flow conditions, repeated sampling during both base flow and storm events would be necessary to more definitively determine the sources of fecal contamination for each watershed.

Open-File Report↗