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Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater
VARMA, M., R. I. FIELD, M. K. STINSON, B. Rukovets, L. J. WYMER, AND R. A. HAUGLAND. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 43(19):4790-4801, (2009).
A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.
1) Determine persistence and degradation rates of genetic markers from different fecal indicator bacterial groups including enterococci, Bacteriodales,and other bacterial groups identified in MCEARD task # 18820, as determined by QPCR analysis, before and after standard wastewater disinfection processes and in ambient surface waters. 2) Determine relationships between reductions of genetic markers from objective 1 and cultivable cells (as determined by standard cultivation methods) among accepted indicator organisms and possibly selected pathogens during and after standard wastewater treatment processes and in ambient surface waters. 3) Evaluate alternative sample preparation procedures and genetic-based analytical methods for their abilities to discriminate between live and dead indicator organisms in wastewater for purpose of selecting most applicable technology for measurement of viable organisms. 4) Subject to outcome of objective 3 studies, determine relationships between degradation rates of genetic markers from total and viable indicator organisms and rates of cultivable cell reductions (as determined by standard culture methods) of currently accepted indicator organisms during and after standard wastewater disinfection processes and in ambient surface waters.
URLs/Downloads:Quantitative Real-time PCR Analysis of Total and Propidium Monoazide-resistant Fecal Indicator Bacteria in Wastewater (PDF,NA pp, 138 KB, about PDF)
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LAB
MICROBIOLOGICAL AND CHEMICAL EXPOSURE ASSESSMENT DIVISION
MICROBIAL EXPOSURE RESEARCH BRANCH