Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains
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. co...
Guardado en:
| Autores principales: | , , , , , |
|---|---|
| Formato: | Articulo |
| Lenguaje: | Inglés |
| Publicado: |
2012
|
| Materias: | |
| Acceso en línea: | http://sedici.unlp.edu.ar/handle/10915/96420 https://ri.conicet.gov.ar/11336/77434 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417533/ https://www.frontiersin.org/articles/10.3389/fcimb.2012.00008/full |
| Aporte de: |
| id |
I19-R120-10915-96420 |
|---|---|
| record_format |
dspace |
| institution |
Universidad Nacional de La Plata |
| institution_str |
I-19 |
| repository_str |
R-120 |
| collection |
SEDICI (UNLP) |
| language |
Inglés |
| topic |
Biología Escherichia coli Shiga-Toxigénica Enterohemorrhagic E. coli Enteropathogenic E. coli Diagnostics E. coli O157 |
| spellingShingle |
Biología Escherichia coli Shiga-Toxigénica Enterohemorrhagic E. coli Enteropathogenic E. coli Diagnostics E. coli O157 Botkin, Douglas J. Galli, Lucía Sankarapani, Vinoth Soler, Michael Rivas, Marta Torres, Alfredo G. Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| topic_facet |
Biología Escherichia coli Shiga-Toxigénica Enterohemorrhagic E. coli Enteropathogenic E. coli Diagnostics E. coli O157 |
| 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. |
| format |
Articulo Articulo |
| author |
Botkin, Douglas J. Galli, Lucía Sankarapani, Vinoth Soler, Michael Rivas, Marta Torres, Alfredo G. |
| author_facet |
Botkin, Douglas J. Galli, Lucía Sankarapani, Vinoth Soler, Michael Rivas, Marta Torres, Alfredo G. |
| author_sort |
Botkin, Douglas J. |
| title |
Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| title_short |
Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| title_full |
Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| title_fullStr |
Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| title_full_unstemmed |
Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains |
| title_sort |
development of a multiplex pcr assay for detection of shiga toxin-producing escherichia coli, enterohemorrhagic e. coli, and enteropathogenic e. coli strains |
| publishDate |
2012 |
| url |
http://sedici.unlp.edu.ar/handle/10915/96420 https://ri.conicet.gov.ar/11336/77434 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417533/ https://www.frontiersin.org/articles/10.3389/fcimb.2012.00008/full |
| work_keys_str_mv |
AT botkindouglasj developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains AT gallilucia developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains AT sankarapanivinoth developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains AT solermichael developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains AT rivasmarta developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains AT torresalfredog developmentofamultiplexpcrassayfordetectionofshigatoxinproducingescherichiacolienterohemorrhagicecoliandenteropathogenicecolistrains |
| bdutipo_str |
Repositorios |
| _version_ |
1764820492110266372 |