title: Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.
abstract: Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.
authors.affiliation: Center for Infectious Diseases, University of Texas School of Public Health, Houston, Texas 77225, USA.
authors.forename: Chase E
authors.initials: CE
authors.lastname: Guion
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Ochoa
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Walker
authors.forename: Francesca
authors.initials: F
authors.lastname: Barletta
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Cleary
mesh_terms: D002648:Child; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D003967:Diarrhea / Q000382:microbiology*; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature; D037521:Virulence Factors / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D037521:Virulence Factors