Search

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta–delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta–delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci — an important consideration in a public health context. Control assay and delta–delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta–delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms. [ABSTRACT FROM AUTHOR]

The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples. [ABSTRACT FROM AUTHOR]

Abstract: A quantitative polymerase chain reaction (qPCR) method for the detection of enterococci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of water quality at recreational beaches. In this study we further examined the applicability of the method for analyses of diverse inland waters as well as tropical marine waters from Puerto Rico based on the frequencies of samples showing presumptive PCR interference. Interference was assessed by salmon DNA sample processing control (SPC) and internal amplification control (IAC) assay analysis results and pre-established acceptance criteria of <3.0 and <1.5 cycle threshold (Ct) offsets from control samples, respectively. SPC assay results were accepted in analyses of 93% of the inland water samples whereas the criterion was met at frequencies of 60% and 97% in analyses of samples from Puerto Rico in two different years of sampling. The functionality of the control assays and their acceptance criteria was assessed on the basis of relative recovery estimates of spiked enterococci target organisms extracted in the presence of water sample filters and sample-free control filters and was supported by observations that recovery estimates from the water sample and control filters were substantially different for samples that failed these criteria. Through the combined use of the SPC and IAC assays, two presumptive types of interference were identified. One type, observed in the tropical marine water samples, appeared to primarily affect the availability of the DNA templates for detection. The second type, observed in river water samples, appeared to primarily affect PCR amplification efficiency. In the presence of DNA template interference, adjustments from SPC assay results by the ΔΔCt comparative Ct calculation method decreased the variability of spiked enterococci recovery estimates and increased the similarity with control filters as compared to unadjusted recovery estimates obtained by the ΔCt calculation method. Use of a higher salmon DNA concentration in the extraction buffer also reduced this type of interference. The effects of amplification interference were largely reversed by dilution of the DNA extracts and even more effectively by the use of an alternative, commercial PCR reagent, designed for the analysis of environmental samples. [Copyright &y& Elsevier]

Abstract: Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×104 target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>103 copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R 2 ≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters. [Copyright &y& Elsevier]

Abstract: Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed ‘pooled’ and ‘mixed’ for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the ‘pooled’ model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the ‘mixed’ model achieved uncertainty estimates similar to the ‘single’ model while increasing the number of available reaction wells per instrument run. [ABSTRACT FROM AUTHOR]

Abstract: Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548cells by QPCR analysis and 1–2499 CFU by MF culture analysis in Lake Michigan and from 8 to 8695cells by QPCR and 3–1941 CFU by MF culture in Lake Erie . Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68. [Copyright &y& Elsevier]

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan™) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure. [Copyright &y& Elsevier]

Fecal pollution remains a challenge for water quality managers at Great Lakes and inland recreational beaches. The fecal indicator of choice at these beaches is typically Escherichia coli (E. coli), determined by culture-based methods that require over 18 h to obtain results. Researchers at the United States Environmental Protection Agency (EPA) have developed a rapid E. coli qPCR methodology (EPA Draft Method C) that can provide same-day results for improving public health protection with demonstrated sensitivity, specificity, and data acceptance criteria. However, limited information is currently available to compare the occurrence of E. coli determined by cultivation and by EPA Draft Method C (Method C). This study provides a large-scale data collection effort to compare the occurrence of E. coli determined by these alternative methods at more than 100 Michigan recreational beach and other sites using the complete set of quantitative data pairings and selected subsets of the data and sites meeting various eligibility requirements. Simple linear regression analyses of composite (pooled) data indicated a correlation between results of the E. coli monitoring approaches for each of the multi-site datasets as evidenced by Pearson R-squared values ranging from 0.452 to 0.641. Theoretical Method C threshold values, expressed as mean log10 target gene copies per reaction, that corresponded to an established E. coli culture method water quality standard of 300 MPN or CFU /100 mL varied only from 1.817 to 1.908 for the different datasets using this model. Different modeling and derivation approaches that incorporated within and between-site variability in the estimates also gave Method C threshold values in this range but only when relatively well-correlated datasets were used to minimize the error. A hypothetical exercise to evaluate the frequency of water impairments based on theoretical qPCR thresholds corresponding to the E. coli water quality standard for culture methods suggested that the methods may provide the same beach notification outcomes over 90% of the time with Method C results differing from culture method results that indicated acceptable and unacceptable water quality at overall rates of 1.9% and 6.6%, respectively. Results from this study provide useful information about the relationships between E. coli determined by culture and qPCR methods across many diverse freshwater sites and should facilitate efforts to implement qPCR-based E. coli detection for rapid recreational water quality monitoring on a large scale in the State of Michigan. • A rapid E. coli qPCR method can provide same-day results for beach monitoring. • A large study compared the occurrence of E. coli by qPCR and culture in Michigan. • Results provide an indication of the degree of correlation between the methods. • Options for a beach notification threshold value for the PCR method are presented. [ABSTRACT FROM AUTHOR]

• NIST SRM® 2917 functions with 13 qPCR assays to fecal waste in samples. • Yeast carrier tRNA stabilizer does not compromise qPCR performance. • All dilution levels exhibited low variability across repeated measurements. • Future use will help minimize variability and make data more comparable across labs. • Material also useful for food production and wastewater surveillance applications. Fecal pollution remains a significant challenge for recreational water quality management worldwide. In response, there is a growing interest in the use of real-time quantitative PCR (qPCR) methods to achieve same-day notification of recreational water quality and associated public health risk as well as to characterize fecal pollution sources for targeted mitigation. However, successful widespread implementation of these technologies requires the development of and access to a high-quality standard control material. Here, we report a single laboratory qPCR performance assessment of the National Institute of Standards and Technology Standard Reference Material 2917 (NIST SRM® 2917), a linearized plasmid DNA construct that functions with 13 recreational water quality qPCR assays. Performance experiments indicate the generation of standard curves with amplification efficiencies ranging from 0.95 ± 0.006 to 0.99 ± 0.008 and coefficient of determination values (R2) ≥ 0.980. Regardless of qPCR assay, variability in repeated measurements at each dilution level were very low (quantification threshold standard deviations ≤ 0.657) and exhibited a heteroscedastic trend characteristic of qPCR standard curves. The influence of a yeast carrier tRNA added to the standard control material buffer was also investigated. Findings demonstrated that NIST SRM® 2917 functions with all qPCR methods and suggests that the future use of this control material by scientists and water quality managers should help reduce variability in concentration estimates and make results more consistent between laboratories. [ABSTRACT FROM AUTHOR]

Sieved vacuum bag dust from the homes of 143 children in Detroit was analyzed by mold specific quantitative PCR (MSQPCR) and the Environmental Relative Moldiness Index (ERMIsm) was calculated for each home. Children living in these homes were grouped as non-asthmatic (n =83), moderately asthmatic (n =28) and severely asthmatic (n =32) based on prescription medication usage for their asthma management (none, occasional and daily, respectively). The mean ERMI for each group of homes was 6.2 for non-asthmatic, 6.3 for moderately asthmatic and 8.2 for severely asthmatic children. The ERMI values in the homes of severely asthmatic children were significantly greater compared to the non-asthmatics (p =0.04 in Wilcoxon Rank-sum test). Aspergillus niger and Aspergillus unguis were the primary mold species that distinguished severely asthmatic children''s homes and non-asthmatic children''s homes (p <0.05; Wilcoxon Rank-sum test). The determination of the home''s ERMI values may aid in prioritizing home remediation efforts, particularly in those children who are at increased risk for asthma exacerbation. [Copyright &y& Elsevier]

Abstract: Using robotics and the MPN technique, a 96-microwell method was developed to compare two procedures for enumeration of viable chlorine-treated B. atrophaeus spores: broth-culture enrichment followed by real-time polymerase chain reaction analysis; and filter plating on agar. Recoveries of chlorine-treated spores were improved by broth enrichment over filter plating, whereas recoveries of non-treated spores were not different in the two procedures. [Copyright &y& Elsevier]

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made. Image 1 • Data QA criteria were established for an EPA E. coli qPCR method (Draft Method C). • QA parameters were slope, intercept for standard curve, LLOQ, Ct values of controls. • Data QA was based on use of prescribed reference and control materials by 21 labs. • The study also provides guidance for labs to establish QA with their own materials. • Polymerase reagent lots should be checked for E. coli signal before use in Method C. [ABSTRACT FROM AUTHOR]

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log 10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials. Image 1 • Variability in E. coli gene copy estimates from an EPA qPCR method was assessed. • Alternative calibration models affected variability but not overall mean estimates. • Failures to meet proposed data quality acceptance criteria excluded 24% of analyses. • Outlier gene copy estimates were not always associated with criteria failures. • Lab experience and their handling of control materials affected method performance. [ABSTRACT FROM AUTHOR]

Highlights • Culture and molecular fecal indicator levels in the wetland remained relatively consistent. • HF-183 signal absent in later wetland treatment stages suggesting treatment effect occurring. • GFD and Campylobacter were detected within the wetland treatment, suggesting avian input. • The only study to use GFD and HF183 MST marker to signal sources of contamination within a wetland. Abstract A constructed, variable-flow treatment wetland was evaluated for its ability to reduce microbial loads from the Banklick Creek, an impacted recreational waterway in Northern Kentucky. For this study, levels of traditional (Escherichia coli and enterococci measured by culture and molecular techniques) and alternative fecal indicators (infectious somatic and F+ coliphage, Clostridium spp. and Clostridium perfringens by culture), potential pathogens (molecular signal of Campylobacter spp.) as well as various microbial source tracking (MST) markers (human fecal marker HF183 and avian fecal marker GFD) were monitored during the summer and early fall through five treatment stages within the Banklick Creek Wetland. No difference in concentrations of traditional or alternative fecal indicators were observed in any of the sites monitored. Microbial source tracking markers were employed to identify sources of fecal contamination within the wetland. Human marker HF183 concentrations at beginning stages of treatment were found to be significantly higher (P value range: 0.0016–0.0003) than levels at later stages. Conversely, at later stages of treatment where frequent bird activity was observed, Campylobacter and avian marker (GFD) signals were detected at significantly higher frequencies (P value range: 0.024 to <0.0001), and both signals were strongly correlated (P = 0.0001). Our study suggests constructed wetlands are an effective means for removal of microbial contamination in ambient waters, but reliance on general fecal indicators is not ideal for determining system efficacy or assessing appropriate remediation efforts. [ABSTRACT FROM AUTHOR]

Abstract An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609.1 and 1611.1 as well as for EPA Draft Method C for Escherichia coli. To obtain the most accurate concentration estimates possible, part of this effort was to develop a data analysis model for determining the fluorescence thresholds that distinguish positive from negative droplets produced by the ddPCR reactions. Versions of this model are described for applications with individual reactions, multiple reactions within a ddPCR system run, and multiple reactions within and across different system runs. The latter version was applied toward determinations of error in the concentration estimates of the standards from replicate analyses of each standard in multiple ddPCR system runs. Mean concentration estimates for the five standards from the ddPCR analyses were 4.356, 3.381, 2.371, 1.641 and 1.071 log 10 copies/5 μL with associated standard deviations of 0.074, 0.082, 0.108, 0.131 and 0.188, respectively. These estimates contrasted with expected log 10 concentrations of 4.6, 3.6, 2.6, 1.9 and 1.3 copies/5 μL, respectively, based on the yield of the plasmid reported by the vendor and spectrophotometric analysis of the initial stock solution of this material. These results illustrate how the analyses of original stocks may lead to potential bias(es) in the concentration estimates of final DNA standards and subsequently in the estimates of unknown test samples determined from these standards in qPCR analyses. Highlights • Plasmid DNA standards were prepared as reference materials for EPA qPCR methods. • The main basis of standards quantification was droplet digital PCR (ddPCR) analysis. • Instrument software & a novel Mixture Model were used to identify positive droplets. • Error estimates from the Mixture Model were used to assess standards stability. • Results illustrate the value of quantifying actual standards over undiluted stocks. [ABSTRACT FROM AUTHOR]

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination. [ABSTRACT FROM AUTHOR]

Recent studies showing an association between fecal indicator organisms (FIOs) in sand and gastrointestinal (GI) illness among beachgoers with sand contact have important public health implications because of the large numbers of people who recreate at beaches and engage in sand contact activities. Yet, factors that influence fecal pollution in beach sand remain unclear. During the 2007 National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study, sand samples were collected at three locations (60 m apart) on weekend days (Sat, Sun) and holidays between June and September at two marine beaches — Fairhope Beach, AL and Goddard Beach, RI — with nearby publicly-owned treatment works (POTWs) outfalls. F + coliphage, enterococci, Bacteroidales , fecal Bacteroides spp., and Clostridium spp. were measured in sand using culture and qPCR-based calibrator-cell equivalent methods. Water samples were also collected on the same days, times and transects as the 144 sand samples and were assayed using the same FIO measurements. Weather and environmental data were collected at the time of sample collection. Mean FIO concentrations in sand varied over time, but not space. Enterococci CFU and CCE densities in sand were not correlated, although other FIOs in sand were. The strongest correlation between FIO density in sand and water was fecal Bacteroides CCE, followed by enterococci CFU, Clostridium spp. CCE, and Bacteroidales CCE. Overall, the factors associated with FIO concentrations in sand were related to the sand–water interface (i.e., sand-wetting) and included daily average densities of FIOs in water, rainfall, and wave height. Targeted monitoring that focuses on daily trends of sand FIO variability, combined with information about specific water quality, weather, and environmental factors may inform beach monitoring and management decisions to reduce microbial burdens in beach sand. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. [ABSTRACT FROM AUTHOR]

Abstract: Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values. [Copyright &y& Elsevier]

Abstract: Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler® II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log10 unit. Platform had the smallest effect, generally exerting <0.1 log10 unit difference in final results. Quantification model led to small differences (0.04–0.2 log10 unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log10 unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples. [Copyright &y& Elsevier]

Abstract: Background: Traditional fecal indicator bacteria (FIB) measurement is too slow (>18 h) for timely swimmer warnings. Objectives: Assess relationship of rapid indicator methods (qPCR) to illness at a marine beach impacted by urban runoff. Methods: We measured baseline and two-week health in 9525 individuals visiting Doheny Beach 2007–08. Illness rates were compared (swimmers vs. non-swimmers). FIB measured by traditional (Enterococcus spp. by EPA Method 1600 or Enterolert™, fecal coliforms, total coliforms) and three rapid qPCR assays for Enterococcus spp. (Taqman, Scorpion-1, Scorpion-2) were compared to health. Primary bacterial source was a creek flowing untreated into ocean; the creek did not reach the ocean when a sand berm formed. This provided a natural experiment for examining FIB-health relationships under varying conditions. Results: We observed significant increases in diarrhea (OR 1.90, 95% CI 1.29–2.80 for swallowing water) and other outcomes in swimmers compared to non-swimmers. Exposure (body immersion, head immersion, swallowed water) was associated with increasing risk of gastrointestinal illness (GI). Daily GI incidence patterns were different: swimmers (2-day peak) and non-swimmers (no peak). With berm-open, we observed associations between GI and traditional and rapid methods for Enterococcus; fewer associations occurred when berm status was not considered. Conclusions: We found increased risk of GI at this urban runoff beach. When FIB source flowed freely (berm-open), several traditional and rapid indicators were related to illness. When FIB source was weak (berm-closed) fewer illness associations were seen. These different relationships under different conditions at a single beach demonstrate the difficulties using these indicators to predict health risk. [Copyright &y& Elsevier]

Abstract: DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method. [Copyright &y& Elsevier]

Mold specific quantitative PCR (MSQPCR) was used to measure the concentrations of the 36 mold species in indoor and outdoor air samples that were taken simultaneously for 48 h in and around 17 homes in Cincinnati, Ohio. The total spore concentrations of 353 per m3 of indoor air and 827 per m3 of outdoor air samples were significantly different (p ≤0.05). However, only the concentrations of Aspergillus penicillioides, Cladosporium cladosporioides types 1 and 2 and Cladosporium herbarum were correlated in indoor and outdoor air samples (p-value≤0.05 and sufficient data for estimate and absolute value rho estimate ≥0.5). These results suggest that interpretation of the meaning of short-term (<48 h) mold measurements in indoor and outdoor air samples must be made with caution. [Copyright &y& Elsevier]

A novel class of strongly fluorescent rhodamine dyes were designed and synthesized by extending the π conjugation of chromophore with limited flexibility. These dyes were shown to have longer absorption in the range of 581 to 631 nm with quantum yields between 0.64 and 0.89. [Copyright &y& Elsevier]

The enzymatic activities of adsorbed Thermomonospora fusca E5 and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. Cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations and adsorption times. Each of these variables is known to have an effect on enzyme structure and activity at an interface. Enzymatic activity was measured after partial elution of the adsorbed layer with both protein-free buffer and the surfactant, dodecyltrimethylammonium bromide. For E5 adsorbed from solution at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% as adsorption time was increased from 0.25 to 24 h. Adsorbed from solution at low concentration (0.001 mg/ml), adsorbed E5 activity decreased by an order of magnitude during a 24 h period. CBHI layers lost activity only after a sufficiently long contact time with the surface, and this effect was not strongly dependent on the enzyme concentration in solution during adsorption. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area. [Copyright &y& Elsevier]