title: Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.
abstract: The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.
authors.affiliation: Southern California Coastal Water Research Project, 3535 Harbor Blvd STE 110, Costa Mesa, CA 92626, USA.
authors.forename: Meredith R
authors.initials: MR
authors.lastname: Raith
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Kelty
authors.forename: John F
authors.initials: JF
authors.lastname: Griffith
authors.forename: Alexander
authors.initials: A
authors.lastname: Schriewer
authors.forename: Stefan
authors.initials: S
authors.lastname: Wuertz
authors.forename: Sophie
authors.initials: S
authors.lastname: Mieszkin
authors.forename: Michele
authors.initials: M
authors.lastname: Gourmelon
authors.forename: Georg H
authors.initials: GH
authors.lastname: Reischer
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Farnleitner
authors.forename: Jared S
authors.initials: JS
authors.lastname: Ervin
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Holden
authors.forename: Darcy L
authors.initials: DL
authors.lastname: Ebentier
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Jay
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Alexandria B
authors.initials: AB
authors.lastname: Boehm
authors.forename: Tiong Gim
authors.initials: TG
authors.lastname: Aw
authors.forename: Joan B
authors.initials: JB
authors.lastname: Rose
authors.forename: E
authors.initials: E
authors.lastname: Balleste
authors.forename: W G
authors.initials: WG
authors.lastname: Meijer
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganesan
authors.forename: Orin C
authors.initials: OC
authors.lastname: Shanks
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D002140:California; D002417:Cattle / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology; D005819:Genetic Markers; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012418:Ruminants / Q000382:microbiology*; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis*
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D012336:RNA, Ribosomal, 16S
keywords: Cattle; Fecal pollution; Microbial source tracking; Ruminants