Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces

Foodborne Pathog Dis. 2012 Jun;9(6):541-8. doi: 10.1089/fpd.2011.1082. Epub 2012 May 8.


An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10⁵ colony-forming units (CFU)/g before enrichment and 10² CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Electrophoresis, Agar Gel
  • Escherichia coli O157 / genetics
  • Escherichia coli O157 / growth & development
  • Escherichia coli O157 / isolation & purification
  • Escherichia coli O157 / metabolism
  • Feces / chemistry
  • Feces / microbiology
  • Genes, Bacterial*
  • Limit of Detection
  • Molecular Typing / methods*
  • Multiplex Polymerase Chain Reaction
  • O Antigens / genetics
  • O Antigens / metabolism*
  • Shiga Toxins / biosynthesis
  • Shiga Toxins / genetics
  • Shiga Toxins / metabolism*
  • Shiga-Toxigenic Escherichia coli / genetics
  • Shiga-Toxigenic Escherichia coli / growth & development
  • Shiga-Toxigenic Escherichia coli / isolation & purification*
  • Shiga-Toxigenic Escherichia coli / metabolism*
  • Species Specificity
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*


  • O Antigens
  • Shiga Toxins
  • Virulence Factors