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Applied and Environmental Microbiology, July 1999, p. 2954-2960, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

John J. Maurer,1,* Denise Schmidt,2 Patricia Petrosko,2 Susan Sanchez,3 Lance Bolton,4 and Margie D. Lee1,3

Departments of Avian Medicine1 and Medical Microbiology and Parasitology,3 University of Georgia, and USDA Agricultural Research Service,4 Athens, Georgia, and Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania2

Received 1 March 1999/Accepted 20 April 1999

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.

* Corresponding author. Mailing address: Department of Avian Medicine, University of Georgia, Athens, GA 30602. Phone: (706) 542-1904. Fax: (706) 542-5630. E-mail: jmaurer{at}calc.vet.uga.edu.

Applied and Environmental Microbiology, July 1999, p. 2954-2960, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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