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.

FIG. 1.

Real-time PCR simultaneously detects eight different diarrheagenic E. coli virulence genes. Data from individual tubes, each containing an EAEC, ETEC, EPEC, STEC, EIEC, or DAEC strain, are shown in a single figure so that the separation between individual amplicon melting curves is illustrated (from left to right: aggR, st, eaeA, lt, stx₁, stx₂, ipaH, and daaD). The y axis (fluorescence) represents the negative derivative of fluorescence over temperature versus temperature.

FIG. 2.

Melting curves of diarrheagenic E. coli. Curves are superimposed for assays of multiple strains of diarrheagenic E. coli to show the reproducibility of given pathotypes. The y axis (fluorescence) represents the negative derivative of fluorescence over temperature versus temperature.

FIG. 3.

Agarose gel of amplicons from the multiplex real-time PCR. The molecular weight ladder is shown in lane 1; strain identification and amplicons are shown in lanes 2 to 7; nonpathogenic E. coli is shown in lane 8.