title: Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.
abstract: 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.
authors.affiliation: Southern California Coastal Water Research Project, 3535 Harbor Blvd Ste 110, Costa Mesa, CA 92626, United States.
authors.forename: Blythe A
authors.initials: BA
authors.lastname: Layton
authors.forename: Yiping
authors.initials: Y
authors.lastname: Cao
authors.forename: Darcy L
authors.initials: DL
authors.lastname: Ebentier
authors.forename: Kaitlyn
authors.initials: K
authors.lastname: Hanley
authors.forename: Elisenda
authors.initials: E
authors.lastname: Ballesté
authors.forename: João
authors.initials: J
authors.lastname: Brandão
authors.forename: Muruleedhara
authors.initials: M
authors.lastname: Byappanahalli
authors.forename: Reagan
authors.initials: R
authors.lastname: Converse
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Farnleitner
authors.forename: Jennifer
authors.initials: J
authors.lastname: Gentry-Shields
authors.forename: Maribeth L
authors.initials: ML
authors.lastname: Gidley
authors.forename: Michèle
authors.initials: M
authors.lastname: Gourmelon
authors.forename: Chang Soo
authors.initials: CS
authors.lastname: Lee
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Lee
authors.forename: Solen
authors.initials: S
authors.lastname: Lozach
authors.forename: Tania
authors.initials: T
authors.lastname: Madi
authors.forename: Wim G
authors.initials: WG
authors.lastname: Meijer
authors.forename: Rachel
authors.initials: R
authors.lastname: Noble
authors.forename: Lindsay
authors.initials: L
authors.lastname: Peed
authors.forename: Georg H
authors.initials: GH
authors.lastname: Reischer
authors.forename: Raquel
authors.initials: R
authors.lastname: Rodrigues
authors.forename: Joan B
authors.initials: JB
authors.lastname: Rose
authors.forename: Alexander
authors.initials: A
authors.lastname: Schriewer
authors.forename: Chris
authors.initials: C
authors.lastname: Sinigalliano
authors.forename: Sangeetha
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Jill
authors.initials: J
authors.lastname: Stewart
authors.forename: Laurie C
authors.initials: LC
authors.lastname: Van De Werfhorst
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Richard
authors.initials: R
authors.lastname: Whitman
authors.forename: Stefan
authors.initials: S
authors.lastname: Wuertz
authors.forename: Jenny
authors.initials: J
authors.lastname: Jay
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Holden
authors.forename: Alexandria B
authors.initials: AB
authors.lastname: Boehm
authors.forename: Orin
authors.initials: O
authors.lastname: Shanks
authors.forename: John F
authors.initials: JF
authors.lastname: Griffith
mesh_terms: D001421:Bacteria, Anaerobic / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002140:California; D004269:DNA, Bacterial / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D062065:Wastewater / Q000382:microbiology; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis*
chemical_list: D004269:DNA, Bacterial; D062065:Wastewater
keywords: Bacteroidales; Bacteroides; Microbial source tracking; Water quality; qPCR