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Quantitative PCR for Genetic Markers of Human Fecal Pollution
Citation:
SHANKS, O. C., C. A. KELTY, M. SIVAGANESAN, M. VARMA, AND R. A. HAUGLAND. Quantitative PCR for Genetic Markers of Human Fecal Pollution. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(17):5507-5513, (2009).
Impact/Purpose:
1) Develop and evaluate qPCR assays and test methods for the detection and quantification of genetic markers from indicator bacteria that are associated with human fecal waste and from two new groups of general fecal indicator bacteria (E. coli and Clostridia) that historically have been widely used or are favored in specific regions 2) Determine the occurrence and densities of genetic markers detected by new qPCR assays developed under objective 1 and compare with occurrence and densities of genetic markers detected by previously developed qPCR assays for enterococci and total Bacterioidalesin waste waters and fecal material from different animal sources. 3) Determine stability of fecal indicator bacteria target DNA sequences in freezer archived filter retentates of ambient surface water samples 4) Determine the densities of human and general fecal indicator markers in a wide range of surface and recreational waters including archived samples from previous NEEAR studies.
Description:
Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. 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. Both assays exhibited a range of quantification from 10 to 1x106 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 was measured in primary effluent wastewater samples collected from 20 geographically distinct locations and compared to quantities estimated by real-time PCR assays specific for ribosomal RNA gene sequences from total Bacteroidales and enterococci 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.