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:

  • Mieszkin, S., Furet, J.-P., Corthier, G., Gourmelon, M. (2009). Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers. Appl. Environ. Microbiol. 75: 3045-3054 [Abstract] [Full Text]  
  • Smith, C. J., Olszewski, A. M., Mauro, S. A. (2009). Correlation of Shiga Toxin Gene Frequency with Commonly Used Microbial Indicators of Recreational Water Quality. Appl. Environ. Microbiol. 75: 316-321 [Abstract] [Full Text]  
  • Rothrock, M. J. Jr, Cook, K. L., Lovanh, N., Warren, J. G., Sistani, K. (2008). Development of a Quantitative Real-Time Polymerase Chain Reaction Assay to Target a Novel Group of Ammonia-Producing Bacteria Found in Poultry Litter. Poult. Sci. 87: 1058-1067 [Abstract] [Full Text]  
  • Toshima, H., Yoshimura, A., Arikawa, K., Hidaka, A., Ogasawara, J., Hase, A., Masaki, H., Nishikawa, Y. (2007). Enhancement of Shiga Toxin Production in Enterohemorrhagic Escherichia coli Serotype O157:H7 by DNase Colicins. Appl. Environ. Microbiol. 73: 7582-7588 [Abstract] [Full Text]  
  • Franz, E., Klerks, M. M., De Vos, O. J., Termorshuizen, A. J., van Bruggen, A. H. C. (2007). Prevalence of Shiga Toxin-Producing Escherichia coli stx1, stx2, eaeA, and rfbE Genes and Survival of E. coli O157:H7 in Manure from Organic and Low-Input Conventional Dairy Farms. Appl. Environ. Microbiol. 73: 2180-2190 [Abstract] [Full Text]  
  • Ravva, S. V., Korn, A. (2007). Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7. Appl. Environ. Microbiol. 73: 2191-2198 [Abstract] [Full Text]  
  • Fukushima, H., Katsube, K., Hata, Y., Kishi, R., Fujiwara, S. (2007). Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR. Appl. Environ. Microbiol. 73: 92-100 [Abstract] [Full Text]  
  • Layton, A., McKay, L., Williams, D., Garrett, V., Gentry, R., Sayler, G. (2006). Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water.. Appl. Environ. Microbiol. 72: 4214-4224 [Abstract] [Full Text]  
  • Hsu, C.-F., Tsai, T.-Y., Pan, T.-M. (2005). Use of the Duplex TaqMan PCR System for Detection of Shiga-Like Toxin-Producing Escherichia coli O157. J. Clin. Microbiol. 43: 2668-2673 [Abstract] [Full Text]  
  • Bono, J. L., Keen, J. E., Miller, L. C., Fox, J. M., Chitko-McKown, C. G., Heaton, M. P., Laegreid, W. W. (2004). Evaluation of a Real-Time PCR Kit for Detecting Escherichia coli O157 in Bovine Fecal Samples. Appl. Environ. Microbiol. 70: 1855-1857 [Abstract] [Full Text]  
  • Chern, E. C., Tsai, Y.-L., Olson, B. H. (2004). Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic Escherichia coli in Agricultural Waste Lagoons. Appl. Environ. Microbiol. 70: 356-362 [Abstract] [Full Text]  
  • Fukushima, H., Tsunomori, Y., Seki, R. (2003). Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools. J. Clin. Microbiol. 41: 5134-5146 [Abstract] [Full Text]  
  • Jinneman, K. C., Yoshitomi, K. J., Weagant, S. D. (2003). Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H- Serotype. Appl. Environ. Microbiol. 69: 6327-6333 [Abstract] [Full Text]  
  • Ibekwe, A. M., Grieve, C. M., Lyon, S. R. (2003). Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent. Appl. Environ. Microbiol. 69: 5060-5069 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»