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Performance Assessment of Human-Specific Microbial Source Tracking Assays End-Point PCR Assays
Citation:
KELTY, C. A., J. C. BLANNON, K. M. WHITE, M. C. MECKES, AND O. C. SHANKS. Performance Assessment of Human-Specific Microbial Source Tracking Assays End-Point PCR Assays. Presented at American Society of Microbiology 108th General Meeting, Boston, MA, June 01 - 05, 2008.
Impact/Purpose:
Poster Presentation
Description:
Waterborne diseases are a significant public health issue, and many originate from contact with water contaminated with human fecal material. Ensuring public water quality requires the use of methods that can rapidly and accurately identify human fecal pollution. We report the performance assessment of five previously published host-specific PCR assays (HF134, HF183, HumM19, HumM22, and B.theta). The goals of this study were to compare assays that either target ribosomal 16S rRNA or chromosomal DNA genes and to identify top performing methods for future characterization in ambient water studies. Each assay was systematically tested with a series of reference samples to establish the limit of detection, sensitivity, specificity, and geographic distribution. For each assay, limit of detection was measured using plasmid derived reference standards. Sensitivity ranged from 2.53x10-10 to 8.2x10-12 g of target DNA with HF183 exhibiting the highest sensitivity and B.theta the lowest. All assays demonstrated high levels of specificity (HumM22, 0.99; HF183, 0.973; B.theta, 0.966; HumM19, 0.947; and HF134, 0.932) when tested against 287 fecal DNA extracts from 20 different animal species. All assays demonstrated a robust spatial distribution (100%) among wastewater samples representing 20 geographically distinct human populations. Data indicated that there is not a clear advantage in sensitivity for PCR assays that target 16S rRNA genes, nor is there an elevated level of specificity with assays that target chromosomal genes. A comparison of performance levels identified the HF183 and HumM22 assays as top candidates for future characterization of genetic marker prevalence in ambient water samples and establishment of potential links between human-specific PCR assays and public health risk.