title: A Four-Plex Real-Time PCR Assay, Based on rfbE, stx1, stx2, and eae Genes, for the Detection and Quantification of Shiga Toxin-Producing Escherichia coli O157 in Cattle Feces.
abstract: Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: Lance W
authors: LW
authors: Noll
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: Pragathi B
authors: PB
authors: Shridhar
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: Xiaorong
authors: X
authors: Shi
authors: 2 Department of Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University , Manhattan, Kansas.
authors: Baoyan
authors: B
authors: An
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: Natalia
authors: N
authors: Cernicchiaro
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: David G
authors: DG
authors: Renter
authors: 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, Kansas.
authors: Tiruvoor G
authors: TG
authors: Nagaraja
authors: 2 Department of Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University , Manhattan, Kansas.
authors: Jianfa
authors: J
authors: Bai
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis* / Q000235:genetics; D000818:Animals; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000235:genetics; D002417:Cattle; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D022622:Shiga Toxin 1 / Q000032:analysis / Q000235:genetics; D022641:Shiga Toxin 2 / Q000032:analysis / Q000235:genetics; D000637:Transaminases / Q000032:analysis / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C073819:eaeA protein, E coli; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase