AUTHOR=Botkin Douglas J., Galli Lucia , Sankarapani Vinoth , Soler Michael , Rivas Marta , Torres Alfredo G.

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=2

YEAR=2012

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2012.00008

DOI=10.3389/fcimb.2012.00008

ISSN=2235-2988

ABSTRACT=<p><italic>Escherichia coli</italic> O157:H7 and other pathogenic <italic>E. coli</italic> 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 <italic>E. coli</italic> (EHEC), Shiga toxin-producing <italic>E. coli</italic> (STEC), and enteropathogenic <italic>E. coli</italic> (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 (<italic>eae</italic>) and long polar fimbriae <italic>(lpfA</italic>) variants, the bundle-forming pilus gene <italic>bfpA</italic>, and the Shiga toxin-encoding genes <italic>stx1</italic> and <italic>stx2</italic>. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (<italic>lpfA1-3</italic>, <italic>lpfA2-2</italic>, <italic>stx1</italic>, <italic>stx2</italic>, and <italic>eae-</italic>γ) and EPEC O127:H6 E2348/69 (<italic>eae-</italic>α, <italic>lpfA1-1</italic>, and <italic>bfpA</italic>) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic <italic>E. coli</italic> 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<sup>4</sup> 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 <italic>E. coli</italic>; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms.</p>