title: Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.
abstract: The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.
authors.affiliation: Department of Civil and Environmental Engineering, University of California Davis, One Shields Ave, Davis, CA 95616, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schriewer
authors.forename: Kelly D
authors.initials: KD
authors.lastname: Goodwin
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Sinigalliano
authors.forename: Annie M
authors.initials: AM
authors.lastname: Cox
authors.forename: David
authors.initials: D
authors.lastname: Wanless
authors.forename: Jakob
authors.initials: J
authors.lastname: Bartkowiak
authors.forename: Darcy L
authors.initials: DL
authors.lastname: Ebentier
authors.forename: Kaitlyn T
authors.initials: KT
authors.lastname: Hanley
authors.forename: Jared
authors.initials: J
authors.lastname: Ervin
authors.forename: Louise A
authors.initials: LA
authors.lastname: Deering
authors.forename: Orin C
authors.initials: OC
authors.lastname: Shanks
authors.forename: Lindsay A
authors.initials: LA
authors.lastname: Peed
authors.forename: Wim G
authors.initials: WG
authors.lastname: Meijer
authors.forename: John F
authors.initials: JF
authors.lastname: Griffith
authors.forename: Jorge
authors.initials: J
authors.lastname: SantoDomingo
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Jay
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Holden
authors.forename: Stefan
authors.initials: S
authors.lastname: Wuertz
mesh_terms: D000818:Animals; D041963:Bacteroidetes / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002140:California; D004269:DNA, Bacterial / Q000145:classification / Q000235:genetics / Q000378:metabolism; D004285:Dogs / Q000382:microbiology*; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces; D012336:RNA, Ribosomal, 16S / Q000145:classification / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Bacteroidales; Canine fecal pollution; Ct; DNQ; FIB; LLOQ; LOD; MST; Microbial source tracking; ND; PCR; Quantitative PCR; ROQ; SIPP; Source Identification Protocol Project; TMDL; cp; cycle threshold; detected but not quantifiable; fecal indicator bacteria; gene copy numbers; limit of detection; lower limit of quantification; microbial source tracking; not detected; polymerase chain reaction; qPCR; quantitative polymerase chain reaction; range of quantification; total maximum daily load