title: Development of a sensitive detection method for stressed E. coli O157:H7 in source and finished drinking water by culture-qPCR.
abstract: A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.
authors.affiliation: Office of Water, Technical Support Center, US Environmental Protection Agency, Cincinnati, Ohio 45268, United States. sen.keya@epa.gov
authors.forename: Keya
authors.initials: K
authors.lastname: Sen
authors.forename: James
authors.initials: J
authors.lastname: L Sinclair
authors.forename: Laura
authors.initials: L
authors.lastname: Boczek
authors.forename: Eugene W
authors.initials: EW
authors.lastname: Rice
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D002713:Chlorine / Q000633:toxicity; D004202:Disinfectants / Q000633:toxicity; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D005618:Fresh Water / Q000737:chemistry / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D013312:Stress, Physiological; D014874:Water Pollutants, Chemical / Q000633:toxicity; D018508:Water Purification; D014881:Water Supply / Q000032:analysis*
chemical_list: D004202:Disinfectants; D014874:Water Pollutants, Chemical; D002713:Chlorine