JCM Accepts, published online ahead of print on 5 March 2008

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J. Clin. Microbiol. doi:10.1128/JCM.02341-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diagnosis of the diarrheagenic Escherichia coli using melting curve analysis of a real-time multiplex polymerase chain reaction

Chase E. Guion, Theresa J. Ochoa, Christopher M. Walker, Francesca Barletta, and Thomas G. Cleary^*

Center for Infectious Diseases, University of Texas School of Public Health, TX; Instituto de Medicina Tropical, Universidad Peruana Cayetano Heredia, Lima, Perú

^* To whom correspondence should be addressed. Email: Thomas.G.Cleary{at}uth.tmc.edu.

The diarrheagenic E. coli are important causes of diarrhea in children from the developing world and now are being recognized as emerging enteropathogens in the developed world. Current methods of diagnosis are too expensive and labor intensive to make it practical to diagnose these organisms routinely. We developed a real-time fluorescence-based multiplex PCR for the diagnosis 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 EAEC, stIa/stIb, and lt for ETEC, eaeA for EPEC and STEC, stx1 and stx2 for STEC, ipaH for EIEC, and daaD for DAEC. Eighty nine of 90 diarrheagenic E. coli and 36/36 non pathogenic E. coli were correctly identified using this approach (specificity 1.00, sensitivity 0.99). The single false negative was a DAEC strain. Total time for preparation of DNA from E. coli colonies on agar plates to completion of PCR and melting curve analysis was less than 90 minutes. The cost of materials was low. Melting point analysis of real time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for diagnosis of diarrheagenic E. coli.

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