|Debroy, C. - PENN STATE|
|Kundrat, J. - PENN STATE|
|Roberts, E. - PENN STATE|
|Davis, M. - PENN STATE|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 21, 2005
Publication Date: August 15, 2005
Citation: Debroy, C., Fratamico, P.M., Briggs, C.E., Kundrat, J., Roberts, E., Liu, Y., Davis, M. 2005. Pcr assays for detection and identification of Escherichia coli serogroups O45 and O55 targeting the wzx and wzy genes in the O antigen gene clusters. Applied and Environmental Microbiology. v. 71. p. 4919-4924. Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and non-harmful E. coli types, 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 are difficult to perform. To develop a more rapid and simple method for detection and typing of the E. coli serogroups O45 and O55, which have caused serious diseases in humans and animals, the DNA sequence of a cluster of genes involved in the production of a specific surface polysaccharide of E. coli O45 was determined, and based on the DNA sequence information for E. coli O45 and similar information already available for E. coli O55, assays called the polymerase chain reaction (PCR), which involve amplification of specific genes in the cluster, were developed for typing of these E. coli serogroups and for detection of the microorganisms in chicken fecal samples. Thus, the use of the E. coli O45- and O55-specific PCR assays facilitates the ability to identify these bacteria, potentially replacing the serotyping procedure.
Technical Abstract: The E. coli O45 O antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (single target), multiplex, and real-time PCR assays were designed targeting the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas, using PCR assays specific for E. coli O55, 97/100 102 strains serotoyped as E. coli O55 were positive for wzx and 98/100 102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 106 and 108 CFU/0.2g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and 055.