Performance of PCR-Based Assays Targeting Bacteroidales Genetic

Jul 21, 2010 - Laboratory, 26 West Martin Luther King Drive, Cincinnati,. Ohio 45268, and U.S. Environmental Protection Agency, Office of Research and...
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Environ. Sci. Technol. 2010, 44, 6281–6288

Performance of PCR-Based Assays Targeting Bacteroidales Genetic Markers of Human Fecal Pollution in Sewage and Fecal Samples O R I N C . S H A N K S , * ,† K A R E N W H I T E , † CATHERINE A. KELTY,† MANO SIVAGANESAN,† JANET BLANNON,† MARK MECKES,† MANJU VARMA,‡ AND RICHARD A. HAUGLAND‡ U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268, and U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268

Received January 28, 2010. Revised manuscript received June 9, 2010. Accepted July 6, 2010.

There are numerous PCR-based assays available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in assay performance. Performance comparisons utilizing feces and raw sewage samples are needed to determine which assays are best suited for expensive and time-consuming field validation, fate, transport, and epidemiology studies. We report the assessment of five end-point PCR and 10 real-time quantitative PCR (qPCR) assays that target genes from presumptive Bacteroidales microorganisms reported to be associated with human feces. Each assay was tested against a reference collection of 54 primary influent sewage samples collected from different geographical locations across the United States and 174 fecal DNA extracts from 23 different animal sources. Experiments indicate that human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest.

Introduction Many methods have been proposed to characterize human fecal pollution in ambient waters using a variety of different microbiology and molecular techniques. Some of the most popular techniques rely on the polymerase chain reaction (PCR) to selectively target a human-associated gene and amplify trace quantities from polluted waters sampled from a beach or watershed. A PCR-based microbial source tracking (MST) method does not only consist of a host-associated assay but also includes a compilation of other protocols for * Corresponding author phone: (513)569-7314; fax: (513)569-7328; e-mail: [email protected]. † National Risk Management Research Laboratory. ‡ National Exposure Research Laboratory. 10.1021/es100311n

Not subject to U.S. Copyright. Publ. 2010 Am. Chem. Soc.

Published on Web 07/21/2010

water sample collection, sample filtration, DNA extraction from filters, estimating sample processing efficiency, monitoring for amplification interference, identifying potential sources of extraneous DNA, and transforming raw data into estimates of DNA target detection frequencies or copy numbers. While all of these steps are vital for a successful MST application and require meticulous validation and performance documentation, the focus of this study is the PCR-based assay only. To date, there are numerous assays available for the detection and/or quantitative assessment of human fecal pollution (1-13). These assays target genes ranging from mitochondrial DNA to rRNA to functional genes involved in microorganism-host interactions. Many of the reported human-associated PCR-based assays target 16S rRNA genes from the Bacteroidales order. PCR amplification of Bacteroidales human-associated genetic markers have been used in many field studies to investigate the sources and levels of fecal pollution at beaches and inland watersheds with variable levels of success (8, 14-16). The conclusions reached in these types of studies can be influenced by a multitude of factors including assay selection. Different Bacteroidales human-associated genetic markers may exhibit different distributions in primary source fecal material from target and nontarget animal sources. However, the performance characterization of genetic marker distributions has been limited, in most cases due to the inclusion of a small number of assays and/or reference fecal and sewage samples in comparison studies (5, 17). For example, the most comprehensive study published to date focusing on human-associated qPCR assays evaluated three assays using reference fecal samples from five nontarget animal sources (5). The determination of genetic marker distribution performance from a larger number of assays across a broader range of human populations and animal sources is needed as a first step toward the implementation of PCR-based MST methods. We describe the performance of five end-point PCR assays (1, 3, 12) and ten qPCR assays (6, 18-22) reported to target Bacteroidales genetic markers associated with human feces. Each assay was tested against a reference collection of primary influent sewage samples representative of 54 different human populations and fecal DNA extracts from 23 known animal sources to estimate assay performance based on the distribution of genetic markers in target and nontarget animal sources. Performance experiments indicate that assay human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest.

Materials and Methods Fecal and Sewage Reference Collections. A series of individual fecal samples (n ) 174) and sewage samples (n ) 54) were collected for use as a reference library of known sources. Fecal samples were collected by rectal grab or immediately after a bowel movement, sealed in a sterile polypropylene container, and stored at -80 °C until DNA extraction (less than 12 months). Individual fecal samples represented a total of 23 different animal sources likely to impact watersheds or beaches including Homo sapiens (human, n ) 16), Anser sp. (Canadian goose; n ) 12), Felis catus (cat, n ) 10), Gallus gallus (chicken, n ) 10), Odocoileus virginianus (white-tail deer, n ) 5), Odocoileus hemionus (mule deer, n ) 5), Cervus VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 6281

elaphus (elk, n ) 5), Alces alces (moose, n ) 1), Antilocapra American (pronghorn, n ) 4), Canis familiariz (dog, n ) 10), Anas sp. (duck, n ) 12), Capra aegagrus (goat, n ) 7), Pelecanus sp. (pelican, n ) 5), Sus scrofa (pig, n ) 22), Laridae (gull, n ) 12), Ovis aries, (sheep, n ) 10), Procyon loter (raccoon, n ) 2), and Meleagris sp. (turkey, n ) 7), Bos taurus (cow, n ) 18), Delphinidae (dolphin, n ) 3), Zalophus californianus (sea lion, n ) 5), and Phocidae (elephant seal, n ) 5). All wildlife samples were collected from feral animals. Each fecal sample was collected from a different individual to maximize the opportunity to observe false positive results. Primary influent sewage samples were collected on-site from 54 different facilities across the United States (Table S1). Facilities were selected based on population served and geographic location. Briefly, 500 mL of untreated influent sewage was collected from each facility and immediately stored on ice. Samples were then packed and shipped on ice overnight to Cincinnati, OH for laboratory testing. Twentyfive milliliters of sewage sample from each sample was filtered through a 0.2 µm pore size Supor-200 filters (Whatman), and each filter was placed in a sterile 1.5 mL microtube and stored at -80 °C until time of DNA extraction (less than 6 months). Total DNA Purification and Quantification. All DNA extractions were performed with the FastDNA Kit for Soils (Q-Biogene, Carlsbad, CA) as described (23). Total DNA extraction yields were determined with a NanoDrop ND1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE). All DNA purifications were diluted to a test concentration of 1 ng/µL per reaction and stored at -20 °C in 50 µL aliquots in GeneMate Slick Low-Adhesion microtubes (ISC BioExpress) until time of analysis. DNA extracts were normalized to a fixed test concentration rather than a mass per gram of feces or liter of sewage to 1) eliminate error introduced by variability in sample consistencies (solid, liquid, or something in between), 2) avoid the need to measure and correct for DNA extraction efficiencies between samples, 3) standardize the test concentration for each DNA extract, and 4) eliminate the potential for differences in DNA concentration between reactions that could impact amplification chemistry. Extraction controls, with purified water substituted for fecal material, were performed each day samples were extracted to monitor for potential extraneous DNA contamination. Primers and Probes. Primer and probe sequences for 15 PCR-based assays reported to be associated with human fecal pollution are reported in Table 1. Human-associated PCRbased assays represent research efforts from five different research laboratories. For human-associated qPCR assays, TaqMan fluorogenic probes were 5′ labeled with 6FAM (6carboxyfluorescein) and 3′ labeled with TAMRA (6-carboxytetramethylrhodamine). The Entero1 multiplex assay was 5′ labeled with TET (tetrachloro derivative of carboxyfluorescein). End-Point PCR Amplification. Five end-point PCR assays were used in this study including HF183, HF134, B.theta, HumM19, and HumM22 as previously reported (Table 1) with the exception that TaKaRa Ex Taq Hot Start Version DNA polymerase (Takara Bio, Inc., Japan) was used for HF134 and HF183 instead of TaKaRa Ex Taq DNA polymerase (1, 3, 12). The Hot Start Version was used to prevent any nonspecific amplification that could occur before samples are loaded into the thermal cycler. Reaction conditions and thermal cycling parameters for all end-point PCR assays are summarized in Table 1. TaKaRa Ex Taq Hot Start Version DNA polymerase, 10XPCR Buffer, and dNTP mixture for PCR reagents were used for HF134, HF183, HumM19, and HumM22 assays. For B.theta, the Eppendorf HotMasterMix 2.5X (Eppendorf, Westbury, NY) was used as previously reported (3). Twenty-five microliter reactions were performed with a DNA Engine Tetrad2 peltier thermal cycler (Bio-Rad, 6282

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Hercules, CA) for 35 cycles. A minimum of two reactions were performed for each DNA extract in 96-well polypropylene plates. Results were visualized by loading 5 µL of PCR product onto either a 2% (HF134, HF183, and B.theta) or 3% (HumM19 and HumM22) agarose gel containing 1X final concentration of GelStar nucleic acid stain (Cambrex BioScience, Rockland, ME). Agarose gels were photographed with a Gel Logic 100 Imaging system (Carestream Health Inc.) and an ultraviolet transilluminator (Fisher Scientific Model: 614A) at the maximum setting with an exposure time of 2.5 s. Unmodified digital images were used to identify positive results. A positive result was defined as the occurrence of the correct size band regardless of intensity. qPCR Amplification. Eleven qPCR assays were used in this study including HF183, HuBac, BsteriF1, BuniF2, BfragF1, PcopriF1, BthetaF2, BvulgF1, HumM2, HumM3, and Entero1 as previously reported (Table 1) with the exception of the HuBac assay. Modifications to the HuBac assay include use of a TAMRA quencher instead of black hole quencher-1 (BHQ1), 7900 HT Fast Real Time Sequence Detector (Applied Biosystems) instead of DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research), and TaqMan Fast Universal Master (Applied Biosytems) instead of QuantiTect PCR Mix (Qiagen, Valencia, CA). All amplifications were performed in a 7900 HT Fast Real Time Sequence Detector (25 µL reaction volume). Reaction conditions and thermal cycling parameters for all qPCR assays are summarized in Table 1. Either TaqMan Fast Universal Master Mix or TaqMan Universal PCR Master Mix reagents were used for all amplifications. All reactions were performed in duplicate in MicroAmp Optical 96-well reaction plates with MicroAmp 96-well Optical Adhesive Film (Applied Biosystems). Data were initially analyzed with Sequence Detector Software (Version 2.2.2), and threshold cycle (CT) values were exported to Microsoft Excel. Monitoring for PCR Inhibition and Other Controls. DNA isolation from fecal and wastewater samples may not remove all substances that can interfere with end-point and qPCR amplification, and the degree of interference may vary between samples. Therefore, an internal amplification control (IAC) designed to evaluate the suitability of isolated DNA for PCR-based analyses was included for each DNA extract. All fecal DNA extracts were screened for inhibition utilizing the Entero1 IAC multiplex assay as previously described (22). The Entero1 IAC assay was designed to simultaneously estimate Enterococcus spp. concentrations and monitor for partial or complete amplification inhibition. The criterion for concluding no significant PCR inhibition of the Entero1 IAC assay was established as a CT ) 34.0 ( 1.5, based on 40 repeated experiments measuring the mean CT values and respective standard deviation (threshold equals two times the standard deviation) for control reactions containing 25 copies of the Entero1 IAC in buffer only. To monitor for potential sources of extraneous DNA during laboratory analyses, a minimum of three no-template amplifications with purified water substituted for template DNA were performed for each 96-well end-point PCR and qPCR experiment. End-Point PCR Specificity, Sensitivity Range, and Prevalence. Presence or absence data generated by duplicate measurements for each individual DNA extract were used to estimate specificity and prevalence. A positive result was scored if either duplicate measurement resulted in the correct sized band at any intensity. The sensitivity range of each PCR assay was determined by testing sewage DNA extracts from 20 randomly selected facilities at five different concentrations including 1 ng, 0.1 ng, 0.01 ng, 1 × 10-3 ng, and 1 × 10-4 ng of total DNA per reaction. In this case, four replicates were tested for each DNA extract dilution and scored as individual data resulting in 80 observations at each

TABLE 1. Primers, Probes, and Reaction Conditionsa

a Platform denotes either an end-point PCR (PCR) or real-time quantitative PCR (qPCR) experiment format. Locus denotes assay gene target. “temp” and “no. of cycles” refer to annealing temperature and number or repeated thermal cycles used for amplification, respectively.

DNA concentration per assay. Sensitivity for each PCR assay at a particular DNA concentration was expressed as the following: sensitivity ) TPC/(TPC+TNI), where TPC represents the total number of samples that tested positive correctly and TNI denotes the total number of amplifications that tested negative incorrectly at a specific total DNA concentration. Prevalence was estimated by testing all 54 sewage samples for the presence of DNA target at a 1 ng/µL of total DNA per reaction and was expressed as the percent of positive detections. Specificity was characterized by testing the 158 nontarget fecal DNA extracts for the presence of target DNA at a 1 ng/µL total DNA per reaction. Specificity was defined as the total number of samples that tested negative correctly (TNC) divided by the sum of TNC and the total number of samples that tested positive incorrectly (TPI) (specificity ) TNC/(TNC+TPI). qPCR Assay Abundance of Genetic Markers in Reference Samples. qPCR assay abundance of DNA targets in human populations was determined by estimating the log10 mean

copy number of DNA targets in sewage samples per 1 ng of total DNA per reaction. Mean nontarget abundance was calculated for each human-associated qPCR assay and was expressed as the sum of log10 mean copy number per 1 ng of total DNA from each animal source composite DNA preparation (see Table 3) divided by the total number of nontarget animal sources (n ) 22). Each animal source composite consisted of equal amounts of DNA from respective individual samples. Calculations and Statistical Analysis. Amplification efficiency (10(1/-slope)/2), a fitted master standard curve coefficient of determination (R-squared value), precision, and range of quantification (ROQ) were calculated from 5 to 21 independently generated standard curves for each humanassociated qPCR assay. The precision of CT measurements was determined from calibration curve DNA standard data and was expressed as a mean percent coefficient of variation calculated from repeated individual measurements at each calibration DNA standard concentration (CV, standard VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. qPCR Calibration Curve Equations and Performance Characteristicsa assay

no. of curves

calibration equation

R-squared

amplification efficiency

ROQ (copies) for target DNA

%CV across ROQ

BfragF1 BthetaF2 BuniF2 BvulgF1 PcopriF1 BsteriF1 HF183 HuBac HumM2 HumM3

8 15 9 6 9 11 11 5 21 21

Y ) 43.8-4.0X Y ) 37.6-3.48X Y ) 39.9-3.52X Y ) 38.4-3.34X Y ) 40.7-3.67X Y ) 42.1-3.40X Y ) 39.9-3.42X Y ) 39.4-3.45X Y ) 42.4-3.73X Y ) 42.5-3.73X

0.961 0.990 0.993 0.991 0.992 0.969 0.987 0.966 0.985 0.985

89% 97% 96% 99.5% 94% 98.5% 98% 97.5% 92.5% 92.5%

10-4 × 104 10-4 × 104 10-4 × 104 10-4 × 104 10-4 × 104 10-4 × 104 10-4 × 104 10-4 × 104 10-1 × 106 10-1 × 106

1.03 2.12 1.24 1.78 2.69 2.73 3.00 2.15 1.44 1.33

a “no. of curves” indicates the number of individual standard curves used to determine a respective master calibration equation. “R-squared” denotes the coefficient of determination representing the proportion of variability in the data set accounted for by the linear model. “amplification efficiency” is equal to 10(1/-slope)/2. “ROQ (copies) for target DNA” refers to the range of quantification measured in copies of target DNA for each respective qPCR assay. “%CV across ROQ” indicates the qPCR precision of measuring standard concentrations expressed as the mean percent coefficient of variation.

deviation expressed as a percentage of the mean). Master calibration curves, unknown DNA copy number estimates, and credible intervals were determined using a Monte Carlo Markov Chain approach (22). This model was selected because it allowed for the estimation of DNA target copy numbers from DNA extracts using a master calibration curve constructed from individual absolute standard curves, while simultaneously propagating uncertainty in estimates from experiment-to-experiment as well as variability between replicate measurements. For a more detailed description of this model, please review Sivaganesan and colleagues (2008). Bayesian calculations were performed using the publicly available software WinBUGS version 1.4.1 (http://www.mrcbsu.cam.ac.uk/bugs) (24). To compare log10 mean copy number estimates between wastewater treatment facilities for each qPCR assay, a two-way nested ANOVA was performed using Statistical Analysis Software (Cary, North Carolina) (25).

Results End-Point PCR Prevalence and Sensitivity Range. Based on analysis of all 54 sewage samples, end-point PCR assays exhibited prevalence values of 100%, 100%, 96.4%, 98.2%, and 96.4% for HF134, HF183, B.theta, HumM19, and HumM22, respectively. The sensitivity for each assay was calculated at five DNA concentrations ranging from 1 ng to 1 × 10-4 ng of total DNA per reaction isolated from sewage samples from 20 different wastewater treatment facilities (Figure 1). qPCR Calibration Curves and Performance Characteristics. Calibration curve equations and performance characteristics of the 10 human-associated qPCR assays are shown in Table 2. ROQs spanned the entire range of standard concentrations tested for all qPCR assays. Precision of CT measurements across defined ROQs for all assays was less than 3% CV and amplification efficiencies ranged from 89% to 112%. Assay Specificity and Abundance of Genetic Markers in Nontarget Animal Sources. Specificity values for end-point PCR assays included 81% (HF134), 95% (HF183), 95% (B.theta), 90% (HumM19), and 95% (HumM22). The abundance of human-associated genetic markers in nonhuman sources was determined for each qPCR assay and ranged from 0.07 to 5.39 log10 copies per 1 ng of total DNA (Table 3). A fixed amount of total DNA (1 ng) was used for all specificity and abundance reactions to standardize measurements. The abundance of genetic markers in nontarget animal hosts ranged from an estimated 0.06 log10 mean copies (HF183) to 2.08 log10 mean copies (HuBac) (Table 3). 6284

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Prevalence and Abundance of Genetic Markers in Human Sources. Prevalence of end-point PCR DNA targets and abundance of qPCR genetic markers in human fecal samples are reported in Table 3. Sewage samples collected from 54 wastewater treatment facilities from 39 different states were used to estimate the distribution of humanassociated genetic markers in human populations. All PCRbased assays yielded prevalence values of 100% for sewage samples at a 1 ng of total DNA per reaction, except BfragF1 (98.7%). A two-way ANOVA comparison of qPCR gene target mean copy number estimates per 1 ng of total DNA in sewage samples indicated no significant difference between 1) BthetaF2 and BfragF1, 2) BuniF2 and HF183, and 3) BsteriF1 and PcopriF1 genetic markers (p > 0.05). A box-and-whisker diagram was used to display differences in human-associated gene targets between sewage sample DNA target estimates for each qPCR assay including the smallest observation, lower quartile (25th percentile), median, upper quartile (75th percentile), largest observation, and outliers (Figure 2). Inhibition and Other Quality Controls. All DNA extracts were diluted with laboratory grade water at least 10-fold to generate 1 ng/µL test concentrations (data not shown). A 10-fold dilution alleviates potential inhibition by 90% and has been shown to effectively remove DNA inhibition from fecal and sewage extracts (17). In addition, the Entero1 qPCR assay was multiplexed with an IAC to monitor for inhibition in all DNA extracts. IAC detection levels indicated the absence of PCR inhibitors in all DNA preparations using an inhibition threshold of 34.0 ( 1.5 CT. No-template controls indicated the absence of extraneous DNA molecules in 100% of control reactions (no false positives of 1024 reactions). All extraction blank controls tested negative.

Discussion Assay Specificity and Distribution of Genetic Markers in Animal Sources. Specificity of end-point PCR assays was estimated based on test results from a reference collection of fecal samples from 158 nontarget individual animals (Table 3). No assay exhibited 100% specificity for human feces suggesting that more than one assay may be needed for the confirmation of human fecal pollution depending on which animals are present in a given watershed or beach environment. Even though no assay was 100% specific, all except one of the end-point assays (HF134) tested demonstrated specificity levels g90% indicating that there are a number of highly specific assays currently available. To evaluate the abundance of human-associated genes in nontarget sources, all qPCR assays were tested against fecal DNA extracts from 22 different nontarget animal sources (Table 3). Mean

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no.

BuniF2 · · · · · · · · · 0.33 ( 0.02 · · · 2.35 ( 0.02 · 2.77 ( 0.02 3.05 ( 0.02 · · · · · 4.28 ( 0.02 0.39

BfragF1

· · · · · · · 1.24 ( 0.1 · · · · · · · 1.44 ( 0.1 2.65 ( 0.1 · 2.31 ( 0.1 2.09 ( 0.1 2.87 ( 0.1 · 3.66 ( 0.1 0.57

· · · · · · · · · · · · · · · 1.66 ( 0.03 3.14 ( 0.03 2.57 ( 0.03 1.30 ( 0.03 · 2.08 ( 0.03 0.74 ( 0.03 3.46 ( 0.03 0.52

BvulgF1 · · · · · · · · · · 1.19 ( 0.05 · · 3.89 ( 0.05 · 1.42 ( 0.05 · 4.38 ( 0.06 4.26 ( 0.06 · · · 4.48 ( 0.06 0.69

PcopriF1 · · · · · · · · · · · · · · · · · 3.31 ( 0.06 4.26 ( 0.07 · · · 4.44 ( 0.07 0.34 · · · 3.47 ( 0.05 0.06

· · · · · · · · · · · · · · · · 0.65 ( 0.05 0.66 ( 0.05

HF183

qPCR assay BsteriF1 · · · · · · · · · 1.82 ( 0.05 · · · · · 2.25 ( 0.05 · · · · · · 3.42 ( 0.05 0.18

HumM2 · · · · · · · · · · · · · · · · 2.83 ( 0.06 · · · · · 3.72 ( 0.06 0.13

HumM3 · · 3.84 ( 0.06 1.20 ( 0.05 1.90 ( 0.05 2.72 ( 0.05 · 2.33 ( 0.05 · 2.29 ( 0.05 2.31 ( 0.05 1.35 ( 0.05 · 2.09 ( 0.05 0.45 ( 0.06 3.64 ( 0.06 4.64 ( 0.06 4.08 ( 0.06 4.23 ( 0.06 2.83 ( 0.05 3.97 ( 0.06 1.84 ( 0.05 5.39 ( 0.07 2.08

HuBac 0.13 ( 0.02 0.45 ( 0.02 · · 0.58 ( 0.02 0.78 ( 0.02 · 0.12 ( 0.02 0.66 ( 0.02 0.74 ( 0.02 · 0.84 ( 0.02 · 0.62 ( 0.02 · 1.43 ( 0.02 1.56 ( 0.02 0.99 ( 0.02 0.07 ( 0.02 · 1.90 ( 0.02 0.42 ( 0.02 3.37 ( 0.02 0.51

BthetaF2

· · · · · · · · · · 5 · · · · · 3 6 · · · 2 15

HF134

· · · · · · · · · · · · · · · · · 1 · · · · 6

· · · · · · · · · · · · · · · · · 2 · · · · ND

B.theta

· · · · · · · · · 1 · · · · · 6 · · · · · · 11

HumM19

end-point PCR assay HF183

· · · · · · · · · · · · · · · · 2 · · · · · 10

HumM22

a ‘no.’ reflects the number of animals tested individually (end-point PCR assays) or as a compsite (qPCR assays). Values listed under qPCR assay columns indicate estimated log10 mean copy number with standard deviation of respective gene target per 1 ng of total DNA from animal source composite. Values listed under end-point PCR assay columns indicate number of individual samples yielding a positive detection to 1 ng of total DNA. A “.” denotes a negative test result. Mean nontarget abundance equals the sum of log10 mean copy estimate for each false positive mean concentration divided by total number of nontarget animal sources (n ) 22). Mean nontarget abundance is not reported for end-point PCR assays. “ND” indicates no data due to discontinuation of Eppendorf HotMasterMix 2.5X reagent during this study.

pronghorn 4 moose 1 mule deer 5 white tail deer 5 Canadian goose 12 duck 12 pelican 5 raccoon 2 gull 12 elk 5 beef cattle 10 dairy cattle 8 goat 7 pig 10 turkey 7 sheep 10 chicken 10 dog 10 cat 10 dolphin 3 sea lion 5 elephant seal 5 human 16 mean nontarget abundance

animal source

TABLE 3. Human-Associated qPCR and PCR Target Abundance and Prevalence in Fecal Sourcesa

FIGURE 1. Sensitivity range for end-point PCR assays. Each point on the plot represents a frequency of positive detection based on 80 amplification reactions. HF183 and HF134 assays target ribosomal rRNA genes and all other assays target nonribosomal genetic markers (see Table 1). abundance in nontarget animal source estimates parsed into two groups including the BfragF1, BvulgF1, PcopriF1, BuniF2, BsteriF1, BthetaF2, HF183, HumM2, and HumM3 assays (2.0 log10 mean copy number) suggesting that most of the qPCR assays tested may exhibit increased levels of specificity after applying corrective models (5, 26) and/or the dilution of low abundance nonhuman fecal derived gene targets in environmental samples. It should be noted that the HuBac qPCR assay was originally reported as 68% specific for human feces based on DNA extracts from 28 reference samples collected from pig (n ) 6), horse (n ) 7), dog (n ) 4), and cattle (n ) 11) where a positive result was defined as an estimated DNA target concentration greater than 1 × 106 copies per gram of feces (6). It is unlikely that the change in thermal cycle instrumentation or reagent supplier are responsible for the

differences between the original report and this study as evidenced by the quantification of DNA targets ranging from 10 to 4 × 104 copies with a high level of precision and amplification efficiency (see Table 2). In addition, the use of a TAMRA quenching molecule instead of a BHQ1 on the HuBac594Bhqf probe (Table 1) is most likely negligible. Previous studies on TAMRA and BHQ1 quenchers suggest that these molecules share similar fluorescence resonance energy transfer quenching efficiencies (27) and that assay specificity is not altered by the use of either quencher (28). Instead, the larger number of fecal samples (174 compared to 28), the inclusion of a more diverse range of animal sources (23 compared to 4), and the absence of a 1 × 106 copies threshold for a positive result are more than likely responsible. Distribution of Host-Associated Genetic Markers in Human Populations. Sewage samples collected from facilities discharging more than 1150 million gallons per day representing fecal material from approximately 6.4 million individuals were used to estimate the distribution of humanassociated genetic markers in United States human populations (Table S1). The high prevalence and abundance of human-associated DNA genetic marker estimates in sewage samples indicate that DNA targets are broadly distributed and may be suitable for use in United States ambient waters. This observation is in contrast to a similar study performed with cattle-associated Bacteroidales genetic markers where dramatic differences were observed between cattle populations originating from different animal feeding facility locations (29). Evaluation of genetic marker distribution in sewage samples also implies that qPCR-based assays targeting 16S rRNA human-associated gene sequences are not always more widely distributed than alternative nonribosomal genetic targets (Figures 1 and 2). In addition, the genetic variation in nonribosomal genes did not always confer increased levels of specificity for end-point PCR assays or decreased abundance of genetic markers in nontarget animal sources for qPCR assays (Table 3). These observations suggest that both ribosomal and alternative genetic markers may be useful for novel MST assay development and that factors such as primer design, assay optimization,

FIGURE 2. Box-and-whisker diagram depicting the relative abundance of gene targets from human associated genetic markers and GenBac3 from all sewage samples collected from all 54 facility locations. Estimated number of gene targets are reported as log10 mean copy number per ng of total DNA. The boundary of the box closest to zero indicates the 25th percentile, the line within the box represents the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 10th and 90th percentiles. A “+” denotes outlier measurements. Assays are presented in order from lowest to highest mean log10 copy numbers per 1 ng of total DNA (right to left). Box plots are the same color if there was no significant difference (p > 0.05) in estimated DNA target copy numbers. Error bars represent the standard deviation of the posterior distribution of the estimated mean log10 target copy number per ng of total DNA. 6286

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and abundance of target organism in pollution source are more significant factors for determining performance, rather than gene locus. State of Science and Implications for Future Research. The primary goal of this study was to characterize the performance of previously reported PCR-based assays targeting genes from Bacteroidales microorganisms based on a series of metrics measured in response to a reference collection of known fecal and sewage sources. Our experiments suggest that the majority of the assays evaluated in this study may be suitable for MST applications. However, some assays clearly performed better than others. While it is evident that some assays such as HuBac and BthetaF2 are not appropriate for discriminating between human and other animal sources of fecal pollution due to high genetic marker abundance in nontarget animal sources (Table 3), it is more difficult to gauge the performance of the remaining assays. The challenge is to determine objectively which performance criteria are the most important. This task proves impossible after considering the vast number of different potential animal sources as well as physical and chemical factors found at a given watershed or beach compared to another. However, there are a number of observations that may prove useful when selecting the appropriate assays for a particular application. HF183 and HumM19 were the only endpoint PCR assays that yielded specificity levels of 95%, prevalence in sewage of 100%, and greater than 10% sensitivity value at the 1 × 10-4 ng of total sewage DNA per reaction concentration. For qPCR assays, only the HF183, BsteriF1, and HumM2 assays exhibited high levels of performance across all criteria including 1) limit of quantification g25 gene copies, 2) %CV across ROQ e 5.0%, 3) fitted calibration curve R2 values g0.95, 4) prevalence in sewage reference samples at 1 ng total DNA per reaction >99%, 5) mean nontarget abundance of genetic marker 3.0 log10 mean copy number. It is important to note that even though these assays performed well with fecal and sewage samples, factors such as the decay rate of genetic markers in environmental matrices and the influence of environmental sample matrix on method sensitivity as well as correlations to general fecal indicators, pathogens, and public health risk will ultimately determine their suitability for water quality and human health risk management applications.

Acknowledgments The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and managed, or partially funded and collaborated in, the research described herein. It has been subjected to the Agency’s peer and administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the author(s) and do not necessarily reflect the official positions and policies of the U.S. EPA. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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Supporting Information Available Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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