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.

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.