title: 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.
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management and Research Laboratory, Cincinnati, Ohio, USA.
authors.forename: Hyatt C
authors.initials: HC
authors.lastname: Green
authors.forename: Richard A
authors.initials: RA
authors.lastname: Haugland
authors.forename: Manju
authors.initials: M
authors.lastname: Varma
authors.forename: Hana T
authors.initials: HT
authors.lastname: Millen
authors.forename: Mark A
authors.initials: MA
authors.lastname: Borchardt
authors.forename: Katharine G
authors.initials: KG
authors.lastname: Field
authors.forename: William A
authors.initials: WA
authors.lastname: Walters
authors.forename: R
authors.initials: R
authors.lastname: Knight
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganesan
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Kelty
authors.forename: Orin C
authors.initials: OC
authors.lastname: Shanks
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012722:Sewage / Q000382:microbiology*; D014871:Water Microbiology; D014876:Water Pollution
chemical_list: D012722:Sewage