Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR

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 t...

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Bibliographic Details
Published in:Journal of Clinical Microbiology 2008-05, Vol.46 (5), p.1752-1757
Main Authors: Guion, Chase E, Ochoa, Theresa J, Walker, Christopher M, Barletta, Francesca, Cleary, Thomas G
Format: Article
Language:English
Subjects:
Bacteriology
Biological and medical sciences
Child
Diarrhea - microbiology
DNA Primers - genetics
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
Escherichia coli
Escherichia coli - classification
Escherichia coli - genetics
Escherichia coli - isolation & purification
Escherichia coli Proteins - genetics
Fundamental and applied biological sciences. Psychology
Humans
Microbiology
Miscellaneous
Polymerase Chain Reaction - economics
Polymerase Chain Reaction - methods
Sensitivity and Specificity
Time Factors
Transition Temperature
Virulence Factors - genetics
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Summary: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), stx₁ and stx₂ 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.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/JCM.02341-07