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United States Department of Agriculture

Agricultural Research Service

Title: DNA Sequence of the Escherichia Coli O103 O Antigen Gene Cluster and Detection of Enterohemorragic E. Coli O103 by Pcr Amplification of the Wzx and Wzy Genes

Authors
item Fratamico, Pina
item Debroy, Chitrita - PENN STATE UNIVERSITY
item Strobaugh, Terence
item Chen, Chin-Yi

Submitted to: Canadian Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 3, 2005
Publication Date: June 1, 2005
Citation: Fratamico, P.M., Debroy, C., Strobaugh Jr, T.P., Chen, C. 2005. Dna sequence of the escherichia coli o103 o antigen gene cluster and detection of enterohemorragic e. coli o103 by pcr amplification of the wzx and wzy genes. Canadian Journal of Microbiology.

Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and non-harmful E. coli types, also referred to as serogroups, also exist. Traditionally, a procedure called serotyping is used to distinguish among the 179 different E. coli serogroups. This procedure relies on the use of antibodies raised in rabbits against different surface components of the bacteria. Serotyping, however, can generally only be performed in specialized laboratories, is labor intensive and may require several days to complete, and one antiserum can react with multiple E. coli serogroups, rendering identification difficult. Thus, due to the lack of simple and rapid methods for detection and identification of harmful and non-harmful E. coli types, the incidence of disease caused by harmful E. coli may be underestimated, and epidemiological studies may be difficult to perform. To develop a more rapid and simple method for detection and typing of the E. coli serogroup O103, an enterohemorrhagic E. coli that has caused serious diseases in humans, the DNA sequence of a cluster of genes involved in the production of a specific surface polysaccharide of the bacterium was determined, and based on the DNA sequence information, an assay called the polymerase chain reaction (PCR), which involves amplification of specific genes in the cluster, was developed for detection and typing of E. coli O103. This strategy can easily also be employed for development of assays for detection and typing of other E. coli serogroups. The use of the E. coli O103-specific PCR assays enhances the ability to identify and type the organism, and the assays can potentially be used to detect E. coli O103 in food, animal, and environmental samples.

Technical Abstract: Methods to detect and distinguish enterohemorrhagic Escherichia coli O103 from other E. coli serogroups are not readily available; therefore, assays based on the polymerase chain reaction (PCR) that are specific for serogroup O103 were developed. To accomplish this, the DNA sequence of the 12,033-bp O antigen gene cluster of Escherichia coli O103 was determined, and 12 open reading frames were identified with all having the same transcriptional direction. Analyses of results indicated that the E. coli O103 wzx (O antigen flippase) and wzy (O antigen polymerase) genes were suitable targets for development of both conventional and real time PCR assays specific for this serogroup. The PCR assays using DNA from 60 E. coli O103 strains, strains representative of non-O103 E. coli serogroups, as well as strains/species of other bacterial genera showed excellent specificity for E. coli O103. Thus, the PCR

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