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PMID: 10388689 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

Applied and environmental microbiology ·Vol. 65 ·No. 7 ·1999-07-00 ·Pages 2954-60

Maurer JJ, Schmidt D, Petrosko P, Sanchez S, Bolton L, Lee MD

Abstract

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.

MeSH Terms
Animals Blotting, Southern Cattle Cosmids/genetics DNA Primers Escherichia coli O157/genetics,isolation & purification,metabolism Feces/microbiology Gene Library Genes, Bacterial Lipopolysaccharides/biosynthesis,immunology Milk/microbiology Molecular Sequence Data O Antigens/biosynthesis Operon Polymerase Chain Reaction/methods Sensitivity and Specificity
Chemicals
DNA Primers Lipopolysaccharides O Antigens
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Maurer J J
Departments of Avian Medicine, University of Georgia, Athens, USA.
Schmidt D
Petrosko P
Sanchez S
Bolton L
Lee M D
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Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/AEM.65.7.2954-2960.1999
Published
1999-07-00
Pages
2954-60
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC91442
Subset
IM
Databases
GENBANK
AF049343
External Links
PubMed source DOI: 10.1128/AEM.65.7.2954-2960.1999 PMC Full Text View journal details
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