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

Autor: Douglas J. Botkin, Marta Rivas, Michael Soler, Alfredo G. Torres, Lucía Galli, Vinoth Sankarapani
Jazyk: angličtina
Rok vydání: 2012
Předmět:
Shiga-toxin producting E. coli
Shiga toxin-producing E. coli
Biología
Enteropathogenic E. coli
lcsh:QR1-502
Ciencias de la Salud
DIAGNOSTICS
medicine.disease_cause
lcsh:Microbiology
Pilus
law.invention
Enteropathogenic Escherichia coli
law
STX2
Genotype
Diagnostics
Escherichia coli Infections
Polymerase chain reaction
Original Research
2. Zero hunger
0303 health sciences
Shiga-Toxigenic Escherichia coli
E. coli O157
Escherichia coli Proteins
3. Good health
STEC
Infectious Diseases
Enterohemorrhagic Escherichia coli
purl.org/becyt/ford/3 [https]
Microbiology (medical)
CIENCIAS MÉDICAS Y DE LA SALUD
Virulence Factors
Immunology
Virulence
Biology
Sensitivity and Specificity
Microbiology
03 medical and health sciences
purl.org/becyt/ford/3.3 [https]
Multiplex polymerase chain reaction
medicine
Humans
Escherichia coli
030304 developmental biology
Intimin
Escherichia coli Shiga-Toxigénica
EPEC
Bacteriological Techniques
Salud Ocupacional
030306 microbiology
ETEC
biochemical phenomena
metabolism
and nutrition
bacterial infections and mycoses
Virology
Food Microbiology
bacteria
Enterohemorrhagic E. coli
Multiplex Polymerase Chain Reaction
Zdroj: CONICET Digital (CONICET)
Consejo Nacional de Investigaciones Científicas y Técnicas
instacron:CONICET
Scopus-Elsevier
SEDICI (UNLP)
Universidad Nacional de La Plata
instacron:UNLP
Frontiers in Cellular and Infection Microbiology, Vol 2 (2012)
Frontiers in Cellular and Infection Microbiology
DOI: 10.3389/fcimb.2012.00008/full
Popis: 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.
Instituto de Genética Veterinaria
Databáze: OpenAIRE
Externí odkaz: