Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains

Authors
Botkin, Douglas J.; Galli, Lucía; Sankarapani, Vinoth; Soler, Michael; Rivas, Marta; Torres, Alfredo G.
Publication Year
2012
Language
English
Format
article
Status
Published version
Description
Escherichia coli O157:H7 and other pathogenic E.coli strains are enteric pathogens associated with food safety threats and which remaina 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 strains 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-gama) and EPEC O127:H6 E2348/69 (eae-alfa, 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 phyogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2×104 CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resultingin 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. 
Fil: Botkin, Douglas J.. University of Texas Medical Branch; Estados Unidos
Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina. University of Texas Medical Branch; Estados Unidos
Fil: Sankarapani, Vinoth. University of Texas Medical Branch; Estados Unidos
Fil: Soler, Michael. University of Texas Medical Branch; Estados Unidos
Fil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina
Fil: Torres, Alfredo G.. University of Texas Medical Branch; Estados Unidos
Subject
STEC
ETEC
EPEC
DIAGNOSTICS
Salud Ocupacional
Ciencias de la Salud
CIENCIAS MÉDICAS Y DE LA SALUD
Access level
Open access
License
https://creativecommons.org/licenses/by-nc/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/77434