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DETECTION AND QUANTIFICATION OF COW FECAL POLLUTION WITH REAL-TIME PCR
Citation:
SHANKS, O. C., E. ATIKOVIC, M. SIVAGANESAN, S. SEIFRING, A. D. BLACKWOOD, J. LU, J. W. SANTO-DOMINGO, R. T. NOBLE, AND R. A. HAUGHLAND. DETECTION AND QUANTIFICATION OF COW FECAL POLLUTION WITH REAL-TIME PCR. Presented at 2007 American Society for Microbiology Conference, Toronto, ON, CANADA, May 21 - 25, 2007.
Impact/Purpose:
to present information
Description:
Assessment of health risk and fecal bacteria loads associated with cow fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for enumeration of two recently described cow-specific genetic markers. Both assays exhibited a range of quantification from 25 to 2x106 copies of target DNA with a coefficient of variation of < 2.1%. One assay can be multiplexed with an internal amplification control to simultaneously detect the cow specific genetic target and presence of amplification inhibitors. Assays only detected cow fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual cow samples (98-100%) collected from five geographically distinct locations. The abundance of each cow-specific genetic marker was measured from 48 individual samples and compared to quantities determined by real-time 16S rRNA PCR assays specific for total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci fecal microorganisms. Assay performances combined with the prevalence of DNA targets across different cattle populations provide experimental evidence for the future application of these quantitative methods in monitoring cow fecal pollution in ambient environmental waters.