AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ibekwe, A. M.
Right arrow Articles by Lyons, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ibekwe, A. M.
Right arrow Articles by Lyons, S. R.
Agricola
Right arrow Articles by Ibekwe, A. M.
Right arrow Articles by Lyons, S. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2002, p. 4853-4862, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4853-4862.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

A. Mark Ibekwe,1* Pamela M. Watt,1 Catherine M. Grieve,1 Vijay K. Sharma,2 and Steven R. Lyons3

George E. Brown Jr. Salinity Laboratory, U.S. Department of Agriculture-Agricultural Research Service, Riverside, California 92507,1 National Animal Disease Center, U.S. Department of Agriculture-Agricultural Research Service, Ames, Iowa 50010,2 Orange County Water District, Fountain Valley, California 927283

Received 21 March 2002/ Accepted 17 July 2002

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the CT and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10-5 pg of E. coli O157:H7 DNA ml-1 equivalent to approximately 6.4 x 103 CFU of E. coli O157:H7 ml-1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >=3.5 x 104 CFU g-1. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g-1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.

* Corresponding author. Mailing address: USDA-ARS-George E. Brown Jr. Salinity Laboratory, 450 W. Big Springs Rd., Riverside, CA 92507. Phone: (909) 369-4828. Fax: (909) 342-4963. E-mail: aibekwe{at}ussl.ars.usda.gov.

Applied and Environmental Microbiology, October 2002, p. 4853-4862, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4853-4862.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals

Copyright © 2002 by the American Society for Microbiology. All rights reserved.