title: Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR.
abstract: Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health  implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food  safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the  O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays  as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence  gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the  assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust  and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.
authors.affiliation: Department of Medical Science, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA. anklamk@svm.vetmed.wisc.edu
authors.forename: Kelly S
authors.initials: KS
authors.lastname: Anklam
authors.forename: Kaushi S T
authors.initials: KS
authors.lastname: Kanankege
authors.forename: Tina K
authors.initials: TK
authors.lastname: Gonzales
authors.forename: Charles W
authors.initials: CW
authors.lastname: Kaspar
authors.forename: Dörte
authors.initials: D
authors.lastname: Döpfer
mesh_terms: D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008461:Meat Products / Q000382:microbiology*; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000302:isolation & purification*; D013997:Time Factors
chemical_list: D022621:Shiga Toxin