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Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources
Citation:
Raith, M. R., C. A. Kelty, J. F. Griffith, A. Schriewer, S. Wuertz, S. Mieszkin, M. Gourmelon, G. H. Reischer, A. H. Farnleitner, J. S. Ervin, P. A. Holden, D. L. Ebentier, J. A. Jay, D. Wang, A. B. Boehm, T. Gim Aw, J. B. Rose, E. Balleste, W. G. Meijer, Mano Sivaganesan, AND O. C. Shanks. Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources . WATER RESEARCH. Elsevier Science Ltd, New York, NY, 47(18):6921-6928, (2013).
Impact/Purpose:
Informs the public on matters relating to the evaluation of molecular methods used to determine fecal sources in environmental waters
Description:
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 cow50 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. Blinded reference challenge 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 total DNA mass, weight mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Experiments indicate that all assays offer precise estimates of respective DNA target concentrations, but show differences in specificity and abundance of genetic markers in non-target fecal pollution sources.
