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Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster
Citation:
SHANKS, O. C., K. M. White, C. A. KELTY, M. SIVAGANESAN, J. C. BLANNON, M. C. MECKES, M. VARMA, AND R. A. HAUGLAND. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster. Presented at American Society for Microbiology 109th General Meeting, Philadelphia, PA, May 17 - 21, 2009.
Impact/Purpose:
To inform the public.
Description:
There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method performance. Laboratory comparisons are needed to determine which assays are best suited for expensive and time consuming fate/transport and epidemiology studies. We describe the performance characterization of 12 assays reported to be specific for human feces. All assays exhibited a range of quantification from 10 to 4x104 copies of target DNA with a coefficient of variation < 3.0%. Each assay was tested against a reference collection of 78 untreated wastewater samples collected from different geographical locations across the United States and 178 fecal DNA extracts from 22 different animal species. qPCR assays demonstrated a broad distribution among untreated wastewater samples (96.8-100%) and indicated specificity levels ranging from 0% to 96%. The abundance of each host-specific genetic marker was measured in both target and non-target animal sources and compared to quantities of enteroccoci determined by real-time 16S rRNA PCR. Comparison experiments indicate that all assays offer precise estimates of respective DNA targets, but show substantial differences in specificity for human feces and abundance of genetic markers in untreated wastewater.
