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Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples
Citation:
Green, H., Rich Haugland, M. Varma, H. Millen, M. Borchardt, K. Field, W. Walters, R. Knight, Mano Sivaganesan, C. Kelty, AND O. Shanks. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 80(10):3086-3094, (2014).
Impact/Purpose:
To inform the public.
Description:
Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protection Agency is planning to conduct a multiple laboratory national validation study to create the first standardized protocol for a human-associated fecal source identification method. In this study, we show that the current top performing TaqMan® HF183 qPCR assay (HF183/BFDrev) routinely forms non-specific amplification products and introduce a modified TaqMan® assay (HF183/BacR287) that alleviates this potential problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, 62 analytical precision, predicted hybridization to a 16S rRNA gene sequences from a reference database, and relative host distributions using fecal and sewage samples. The modified HF183/BacR287 assay either maintains or improves upon the positive attributes of the original HF183/BFDrev assay. In addition, safeguards against amplification inhibition are introduced including the use of a proprietary qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control. In light of the expanding use of PCR-based methods that rely on the detection of extremely small concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan® HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.
