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United States Department of Agriculture

Agricultural Research Service


item Bono, James
item Keen, James
item Clawson, Michael
item Heaton, Michael
item Fox, James
item Chitko Mckown, Carol
item Laegreid, William

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2004
Publication Date: May 1, 2004
Citation: Bono, J.L., Keen, J.E., Clawson, M.L., Heaton, M.P., Fox, J.M., Chitko McKown, C.G., Laegreid, W.W. 2004. Discrimination of Escherichia coli O157:H7 from other bacteria by genotyping single nucleotide polymorphisms [abstract]. American Society for Microbiology. Paper No. C-311.

Technical Abstract: E. coli O157:H7 is the major cause of hemorrhagic colitis and hemolytic uremic syndrome in the United States. Rapid detection of E. coli O157:H7 is important so that infection control interventions can be instituted to minimize transmission, contamination, and disease by this pathogen. A number of genes have been targeted for E. coli O157:H7 diagnostic PCR, but many genes are not exclusively diagnostic for E. coli O157:H7 because other bacteria may have one or more of these genes. For example, several PCR targets have been designed against genes from the E. coli O157 O-antigen operon that had high sensitivity and specificity for E. coli O157. However, these same PCR targets were not able to distinguish between shiga toxin containing E. coli O157:H7 and non-shiga toxin containing E. coli O157:non-H7 isolates. We hypothesized the existence of genomic differences in the E. coli O157 O-antigen operon between the E. coli O157:H7 and non-E. coli O157:non-H7 that could be useful to design a PCR test specific for E. coli O157:H7. The per and wbdR genes from the E. coli O157 O-antigen synthesis operon were sequenced from 18 E. coli O157:H7 isolates and 8 non-E. coli O157:H7 isolates. A single nucleotide polymorphism (SNP) from both genes, per-sp11 and wbdR-sp13, defined two alleles with E. coli O157:H7 isolates belonging to a group with allele A and E. coli O157:non-H7 isolates belonging to a group with allele B. Real-time PCR genotyping assays were designed against the two SNPs and tested against 147 E. coli O157:H7, 33 E. coli O157:non-H7, and 123 non-E. coli O157 isolates. Two Graph-Receiver Operator Characteristic (TG-ROC) analysis of cycle threshold (CT) values from the 303 isolates indicated an optimal CT cutoff value of 32.0 for wbdR-sp13 and 35.6 for per-sp11. Using these cutoff values, the wbdR-sp13 assay had a 100% sensitivity and specificity, while the per-sp11 assay had 99% sensitivity and specificity for the detection of E. coli O157:H7. These results suggest that per-sp11 and wbdR-wp13 are excellent candidates for use as targets in real-time PCR assays to quickly detect E. coli O157:H7.

Last Modified: 7/27/2016
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