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United States Department of Agriculture

Agricultural Research Service

Title: Real-Time Pcr Assays for the Quantitation of Enteropathogenic E.COLI in Water

Authors
item Karns, Jeffrey
item Shelton, Daniel

Submitted to: Applied and Environmental Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2003
Publication Date: N/A

Technical Abstract: Testing the quality of water usually involves determining the number of fecal microorganisms by culturing in or on various microbiological media. While these tests are useful for determining the likelihood that water has been contaminated by fecal material they do not give a true indication of actual pathogenic organisms in the sample. The purpose of this study was to develop real-time PCR (RT-PCR) assays for the quantitative assessment of potentially enteropathogenic E. coli as well as total E.coli in water. Total E. coli are quantified using a TaqMan primer/probe set specific for the lacZ gene of E. coli. This assay successfully detected 47 of 47 strains of E. coli representing 13 O-serotypes and 3 wild-type strains. A commercial plasmid containing the lacZ gene of E. coli is used as a copy number standard for quantitation. Enteropathogenic strains of E. coli (EPEC) are quantified using one of two TaqMan primer/probe sets that target genes in the locus of enterocyte effacement (LEE) on the chromosome. The eae set targets a conserved region of eaeA which encodes intimin. This primer/probe set detected 40 of 40 EPEC and enterohemorrhagic (EHEC) strains representing 10 O-serotypes but did not detect 4 non-EPEC clinical isolates (3 serotypes), wild-type strains, or Citrobacter freundii. The tir primer/probe set targets a variable region of the gene encoding the translocated intimin receptor; it detected only O157, O55, and O125 strains. For both the eae and tir assays a constructed plasmid containing small portions of the target genes is used as a copy number standard. Comparing the ratio of eaeA or tir gene copies to lacZ gene copies allows the assessment of the portion of the total population of E. coli in a water sample that are enteropathogenic. Since the population of E. coli in most water samples is too low for direct detection, the RT-PCR can be performed on DNA isolated from enrichment cultures. If all strains of E. coli are presumed to grow at similar rates in enrichments and, if a total E. coli count is done by conventional culture, this ratio can be used to quantify low levels of EPEC and EHEC strains in water.

Last Modified: 8/27/2014
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