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Journal of Clinical Microbiology, September 2004, p. 4092-4100, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4092-4100.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic Escherichia coli

Udo Reischl,1* Mohammad T. Youssef,2,3 Hans Wolf,1 Eija Hyytia-Trees,3 and Nancy A. Strockbine3

Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany,1 Department of Biological Sciences, Yarmouk University, Irbid, Jordan,2 National Escherichia coli-Shigella Reference Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia3

Received 1 December 2003/ Returned for modification 16 March 2004/ Accepted 27 May 2004

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

* Corresponding author. Mailing address: Institute of Medical Microbiology and Hygiene, University Hospital of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Phone: 49-941-944-6450. Fax: 49-941-944-6402. E-mail: Udo.Reischl{at}klinik.uni-regensburg.de.

Journal of Clinical Microbiology, September 2004, p. 4092-4100, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4092-4100.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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