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Applied and Environmental Microbiology, September 2009, p. 5507-5513, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00305-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Quantitative PCR for Genetic Markers of Human Fecal Pollution{triangledown}

Orin C. Shanks,1* Catherine A. Kelty,1 Mano Sivaganesan,1 Manju Varma,2 and Richard A. Haugland2

U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268,1 U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 452682

Received 6 February 2009/ Accepted 2 July 2009

Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 x 106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

* Corresponding author. Mailing address: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45268. Phone: (513) 569-7314. Fax: (513) 569-7328. E-mail: shanks.orin{at}epa.gov

{triangledown} Published ahead of print on 10 July 2009.

Applied and Environmental Microbiology, September 2009, p. 5507-5513, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00305-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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