title: 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.
abstract: 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.
authors.affiliation: Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas 66506-5606, USA. jbai@vet.k-state.edu
authors.forename: Jianfa
authors.initials: J
authors.lastname: Bai
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Paddock
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Shi
authors.forename: Shubo
authors.initials: S
authors.lastname: Li
authors.forename: Baoyan
authors.initials: B
authors.lastname: An
authors.forename: Tiruvoor G
authors.initials: TG
authors.lastname: Nagaraja
mesh_terms: D000818:Animals; D002417:Cattle; D004587:Electrophoresis, Agar Gel; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism; D005243:Feces / Q000737:chemistry / Q000382:microbiology; D005798:Genes, Bacterial; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction; D019081:O Antigens / Q000235:genetics / Q000378:metabolism*; D022603:Shiga Toxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism*; D013045:Species Specificity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D019081:O Antigens; D022603:Shiga Toxins; D037521:Virulence Factors