Development of PCR assays targeting genes in O-antigen gene clusters for detection and identification of Escherichia coli O45 and O55 serogroups

Abstract

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in 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 by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 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 10(6) and 10(8) CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.

FIG. 1.

Multiplex PCRs targeting the E. coli O45 wzx and wzy (A) and O55 wzx and wzy (B) genes. (A) Lane M, molecular weight markers; lane 1, E. coli O45 (positive control) (multiplex PCR showing the amplified wzx [255 bp] and wzy [451 bp] genes); lane 2, E. coli K-12 (negative control); lane 3, results from a chicken fecal sample spiked with E. coli O45 at 10⁶ CFU/0.2 g; lane 4, result from a chicken fecal sample spiked with E. coli O45 at 10⁸ CFU/0.2 g. (B) Lane M, molecular weight markers; lane 1, E. coli O55 (positive control) (multiplex PCR showing the amplified wzx [683 bp] and wzy [147 bp] genes); lane 2, E. coli K-12, (negative control); lane 3, results from a chicken fecal sample spiked with E. coli O55 at 10⁶ CFU/0.2 g; lane 4, results from a chicken fecal sample spiked with E. coli O55 at 10⁸ CFU/0.2 g.