Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

ABSTRACT Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

[1]  Mano Sivaganesan,et al.  Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples. , 2010, Environmental science & technology.

[2]  Julie Kinzelman,et al.  Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria. , 2012, Environmental science & technology.

[3]  Daniel E. Williams,et al.  Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water , 2006, Applied and Environmental Microbiology.

[4]  A. J. Jones,et al.  At Least 1 in 20 16S rRNA Sequence Records Currently Held in Public Repositories Is Estimated To Contain Substantial Anomalies , 2005, Applied and Environmental Microbiology.

[5]  W. Ludwig,et al.  SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB , 2007, Nucleic acids research.

[6]  J. Hoorfar,et al.  Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays , 2004, Journal of Clinical Microbiology.

[7]  T. Gardner,et al.  Evaluation of Bacteroides markers for the detection of human faecal pollution , 2007, Letters in applied microbiology.

[8]  A. Goonetilleke,et al.  Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh. , 2010, Canadian journal of microbiology.

[9]  K. M. Ritalahti,et al.  Design and application of an internal amplification control to improve Dehalococcoides mccartyi 16S rRNA gene enumeration by qPCR. , 2013, Environmental science & technology.

[10]  Dan Wang,et al.  Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods. , 2013, Water research.

[11]  S. Weisberg,et al.  Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters , 2012, Journal of applied microbiology.

[12]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[13]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[14]  K. Gibson,et al.  Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples. , 2012, Water research.

[15]  Orin C. Shanks,et al.  Distribution of Genetic Marker Concentrations for Fecal Indicator Bacteria in Sewage and Animal Feces , 2012, Applied and Environmental Microbiology.

[16]  Marion W. Jenkins,et al.  Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays. , 2009, Water research.

[17]  W. Meijer,et al.  Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union. , 2007, Water research.

[18]  Willy Verstraete,et al.  Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater. , 2005, Environmental microbiology.

[19]  Orin C. Shanks,et al.  Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study. , 2013, Water research.

[20]  Mano Sivaganesan,et al.  Quantitative PCR for Genetic Markers of Human Fecal Pollution , 2009, Applied and Environmental Microbiology.

[21]  Rolf Backofen,et al.  TassDB: a database of alternative tandem splice sites , 2006, Nucleic Acids Res..

[22]  Mano Sivaganesan,et al.  A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards , 2008, BMC Bioinformatics.

[23]  Jiyoung Lee,et al.  Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination. , 2010, Journal of microbiological methods.

[24]  K. Field,et al.  Sensitive detection of sample interference in environmental qPCR. , 2012, Water research.

[25]  Dan Wang,et al.  Performance of forty-one microbial source tracking methods: a twenty-seven lab evaluation study. , 2013, Water research.

[26]  Katharine G. Field,et al.  Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes , 2000, Applied and Environmental Microbiology.

[27]  Orin C. Shanks,et al.  Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon , 2006, Applied and Environmental Microbiology.

[28]  Orin C. Shanks,et al.  Combining land use information and small stream sampling with PCR-based methods for better characterization of diffuse sources of human fecal pollution. , 2011, Environmental science & technology.

[29]  Mary E. Schoen,et al.  Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters , 2012, Applied and Environmental Microbiology.

[30]  R. Haugland,et al.  Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method. , 2012, Water research.

[31]  Alimuddin Zumla,et al.  Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon , 2008, BMC Research Notes.

[32]  B. Gilpin,et al.  Source Tracking in Australia and New Zealand: Case Studies , 2011 .

[33]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[34]  S. Okabe,et al.  Quantification of host-specific Bacteroides–Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater , 2007, Applied Microbiology and Biotechnology.

[35]  A. Farnleitner,et al.  A quantitative real‐time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area , 2007, Letters in applied microbiology.

[36]  Orin C. Shanks,et al.  Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR. , 2010, Systematic and applied microbiology.

[37]  S. Wuertz,et al.  16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: a Bayesian approach. , 2007, Water research.

[38]  Katharine G. Field,et al.  A PCR Assay To Discriminate Human and Ruminant Feces on the Basis of Host Differences in Bacteroides-Prevotella Genes Encoding 16S rRNA , 2000, Applied and Environmental Microbiology.

[39]  Orin C. Shanks,et al.  Differential decay of human faecal Bacteroides in marine and freshwater. , 2011, Environmental microbiology.

[40]  Christian L. Lauber,et al.  PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers , 2011, Bioinform..

[41]  J. Larrick,et al.  Competitive PCR , 1992, Nature.

[42]  J. Trevors,et al.  Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers , 2010, Applied Microbiology and Biotechnology.