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Development of a multiplex PCR assay for detection of Shiga toxin-producing Escherichia coli, enterohemorrhagic E. coli, and enteropathogenic E. coli strains. 2012

Douglas J Botkin, and Lucía Galli, and Vinoth Sankarapani, and Michael Soler, and Marta Rivas, and Alfredo G Torres
Department of Microbiology and Immunology, University of Texas Medical Branch Galveston, TX, USA.

Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain's respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 10(4) CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms.

UI MeSH Term Description Entries
D004927 Escherichia coli Infections Infections with bacteria of the species ESCHERICHIA COLI. E coli Infections,E. coli Infection,Infections, E coli,Infections, Escherichia coli,E coli Infection,E. coli Infections,Escherichia coli Infection,Infection, E coli,Infection, E. coli,Infection, Escherichia coli
D005516 Food Microbiology The presence of bacteria, viruses, and fungi in food and food products. This term is not restricted to pathogenic organisms: the presence of various non-pathogenic bacteria and fungi in cheeses and wines, for example, is included in this concept. Microbiology, Food
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001431 Bacteriological Techniques Techniques used in studying bacteria. Bacteriologic Technic,Bacteriologic Technics,Bacteriologic Techniques,Bacteriological Technique,Technic, Bacteriological,Technics, Bacteriological,Technique, Bacteriological,Techniques, Bacteriological,Bacteriologic Technique,Bacteriological Technic,Bacteriological Technics,Technic, Bacteriologic,Technics, Bacteriologic,Technique, Bacteriologic,Techniques, Bacteriologic
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D054308 Enteropathogenic Escherichia coli Strains of ESCHERICHIA COLI characterized by attaching-and-effacing histopathology. These strains of bacteria intimately adhere to the epithelial cell membrane and show effacement of microvilli. In developed countries they are associated with INFANTILE DIARRHEA and infantile GASTROENTERITIS and, in contrast to ETEC strains, do not produce ENDOTOXINS. E coli, Enteropathogenic,EPEC,Escherichia coli, Enteropathogenic,Enteropathogenic E. coli
D054323 Shiga-Toxigenic Escherichia coli Strains of ESCHERICHIA COLI with the ability to produce at least one or more of at least two antigenically distinct, usually bacteriophage-mediated cytotoxins: SHIGA TOXIN 1 and SHIGA TOXIN 2. These bacteria can cause severe disease in humans including bloody DIARRHEA and HEMOLYTIC UREMIC SYNDROME. E coli, Verotoxigenic,Escherichia coli, Verotoxigenic,STEC,Verotoxigenic Escherichia coli,Shiga Toxigenic E. coli,Shiga Toxigenic Escherichia coli,Shiga Toxin-Producing Escherichia coli,VTEC,Vero Cytotoxin-Producing Escherichia coli,Verotoxigenic E. coli,Verotoxin-Producing Escherichia coli
D054324 Enterohemorrhagic Escherichia coli Strains of ESCHERICHIA COLI that are a subgroup of SHIGA-TOXIGENIC ESCHERICHIA COLI. They cause non-bloody and bloody DIARRHEA; HEMOLYTIC UREMIC SYNDROME; and hemorrhagic COLITIS. An important member of this subgroup is ESCHERICHIA COLI O157-H7. E coli, Enterohemorrhagic,EHEC,Escherichia coli, Enterohemorrhagic,Enterohemorrhagic E coli,Enterohemorrhagic E. coli
D060885 Multiplex Polymerase Chain Reaction Methods for using more than one primer set in a polymerase chain reaction to amplify more than one segment of the target DNA sequence in a single reaction. Multiplex Ligation-Dependent Probe Amplification,Multiplex PCR,Triplex PCR,Triplex Polymerase Chain Reaction,Multiplex Ligation Dependent Probe Amplification,PCR, Multiplex,PCR, Triplex
D029968 Escherichia coli Proteins Proteins obtained from ESCHERICHIA COLI. E coli Proteins

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