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Comparison of PCR-Based Assays for the Characterization of Cattle Fecal Pollution in California
Citation:
KELTY, C. A., M. SIVAGANESAN, O. C. SHANKS, M. Raith, J. Griffith, A. Schriewer, S. Wuertz, S. Mieszkin, M. Guormelon, G. Reischer, A. Farnleitner, J. Ervin, T. Holden, J. Jay, AND A. Boehm. Comparison of PCR-Based Assays for the Characterization of Cattle Fecal Pollution in California. Presented at American Society of Microbiologist 112th General Meeting, San Francisco, CA, June 16 - 19, 2012.
Impact/Purpose:
To inform the public.
Description:
The state of California has mandated the production of a guidance document on the application of microbial source tracking 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 the State of California. 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 64 filters (32 duplicate samples) containing fecal pollution from 12 different animal sources suspected to impact water quality in California recreational waters. Blinded filters contained either a single fecal source or a mixture of two different sources. Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target animal sources. 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 DNA template mass, as well as Bacteroidales, E. coli, and enterococci determined by 16S rRNA qPCR and culture-based approaches (E. coli and enterococci only). Experiments indicate that all assays offer precise estimates of respective DNA targets, but show differences in specificity and abundance of genetic markers in non-target fecal pollution sources.
