|Green, Hyatt -|
|Haugland, Richard -|
|Varma, Manju -|
|Millen, Hana -|
|Field, Katherine -|
|Walters, William -|
|Knight, R. -|
|Kelty, Catherine -|
|Shanks, Orin -|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2014
Publication Date: May 1, 2014
Citation: Green, H.C., Haugland, R.A., Varma, M., Millen, H.T., Borchardt, M.A., Field, K.G., Walters, W.A., Knight, R., Kelty, C.A., Shanks, O.C. 2014. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples. Applied and Environmental Microbiology. 80:3086-3094. Interpretive Summary: Fecal pollution of the nation’s waters can stem from three major sources, wildlife, livestock, and humans. A key first step in reducing pollution levels is identifying the source. Human fecal waste in water is most commonly identified by detecting a type of bacteria within the group called Bacteroides that are only found in the human gastrointestinal system. The tests for these bacteria are advanced in that they are genetic based; however, many similar genes exist in the environment leading to ambiguous test results. This study modified several procedures of the most common test, improving its overall performance and ability to distinguish human fecal wastes from other sources. Methods derived from this study will be included by the U.S. Environmental Protection Agency in a national validation study to create the first standardized protocol for human-associated fecal source identification.
Technical Abstract: Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. 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, partially based on these methods. In this study, we show that a current TaqMan® HF183 qPCR assay (HF183/BFDrev) routinely forms non-specific amplification products, and we introduce a modified TaqMan® assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative host distributions in 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.