Geology topics

Patricia A. Holden

Publications and source records attributed to Patricia A. Holden.

4 recordsLinked to original sources

USGS2014

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Journal of Environmental Management↗

USGS2013

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources

Here we report results from a multi-laboratory ( n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR ® Green qPCR method, and two TaqMan ® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.

Water Research↗

USGS2013

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

Water Research↗

USGS2012

Sources of fecal indicator bacteria to groundwater, Malibu Lagoon and the near-shore ocean, Malibu, California, USA

Onsite wastewater treatment systems (OWTS) used to treat residential and commercial sewage near Malibu, California have been implicated as a possible source of fecal indicator bacteria (FIB) to Malibu Lagoon and the near-shore ocean. For this to occur, treated wastewater must first move through groundwater before discharging to the Lagoon or ocean. In July 2009 and April 2010, δ 18 O and δD data showed that some samples from water-table wells contained as much as 70% wastewater; at that time FIB concentrations in those samples were generally less than the detection limit of 1 Most Probable Number (MPN) per 100 milliliters (mL). In contrast, Malibu Lagoon had total coliform, Escherichia coli , and enterococci concentrations as high as 650,000, 130,000, and 5,500 MPN per 100 mL, respectively, and as many as 12% of samples from nearby ocean beaches exceeded the U.S. Environmental Protection Agency single sample enterococci standard for marine recreational water of 104 MPN per 100 mL. Human-associated Bacteroidales , an indicator of human-fecal contamination, were not detected in water from wells, Malibu Lagoon, or the near-shore ocean. Similarly, microarray (PhyloChip) data show Bacteroidales and Fimicutes Operational Taxanomic Units (OTUs) present in OWTS were largely absent in groundwater; in contrast, 50% of Bacteroidales and Fimicutes OTUs present in the near-shore ocean were also present in gull feces. Terminal-Restriction Length Fragment Polymorphism (T-RFLP) and phospholipid fatty acid (PLFA) data showed that microbial communities in groundwater were different and less abundant than communities in OWTS, Malibu Lagoon, or the near-shore ocean. However, organic compounds indicative of wastewater (such as fecal sterols, bisphenol-A and cosmetics) were present in groundwater having a high percentage of wastewater and were present in groundwater discharging to the ocean. FIB in the near-shore ocean varied with tides, ocean swells, and waves. Movement of water from Malibu Lagoon through the sand berm at the mouth of the Lagoon contributed FIB to the adjacent beach at low tide. Similar increases in FIB concentrations did not occur at beaches adjacent to unsewered residential development, although wastewater indicator compounds and radon-222 (indicative of groundwater discharge) were present. High FIB concentrations at high tide were not related to groundwater discharge, but may be related to FIB associated with debris accumulated along the high-tide line.

California↗