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.
Copyright © 2013. Published by Elsevier Ltd.