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Journal of Clinical Microbiology, May 2008, p. 1752-1757, Vol. 46, No. 5
0095-1137/08/$08.00+0     doi:10.1128/JCM.02341-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Chase E. Guion,1 Theresa J. Ochoa,1,2 Christopher M. Walker,1 Francesca Barletta,2 and Thomas G. Cleary1*

Center for Infectious Diseases, University of Texas School of Public Health, Houston, Texas,1 Instituto de Medicina Tropical, Universidad Peruana Cayetano Heredia, Lima, Peru2

Received 5 December 2007/ Returned for modification 9 February 2008/ Accepted 22 February 2008

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.

* Corresponding author. Mailing address: University of Texas School of Public Health, Center for Infectious Diseases, P.O. Box 20186, Houston, TX 77225. Phone: (713) 500-5714. Fax: (713) 500-5688. E-mail: Thomas.G.Cleary{at}uth.tmc.edu

{triangledown} Published ahead of print on 5 March 2008.

Journal of Clinical Microbiology, May 2008, p. 1752-1757, Vol. 46, No. 5
0095-1137/08/$08.00+0     doi:10.1128/JCM.02341-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ochoa, T. J., Ruiz, J., Molina, M., Del Valle, L. J., Vargas, M., Gil, A. I., Ecker, L., Barletta, F., Hall, E., Cleary, T. G., Lanata, C. F. (2009). High Frequency of Antimicrobial Drug Resistance of Diarrheagenic Escherichia coli in Infants in Peru. Am J Trop Med Hyg 81: 296-301 [Abstract] [Full Text]  
  • Barletta, F., Ochoa, T. J., Ecker, L., Gil, A. I., Lanata, C. F., Cleary, T. G. (2009). Validation of Five-Colony Pool Analysis Using Multiplex Real-Time PCR for Detection of Diarrheagenic Escherichia coli. J. Clin. Microbiol. 47: 1915-1917 [Abstract] [Full Text]  


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