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Journal of Clinical Microbiology, July 2002, p. 2555-2565, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2555-2565.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

Udo Reischl,1* Mohammad T. Youssef,2,3 Jochen Kilwinski,4 Norbert Lehn,1 Wen Lan Zhang,5 Helge Karch,5 and Nancy A. Strockbine2

Institute of Medical Microbiology and Hygiene, University of Regensburg, D-93053 Regensburg,1 State Laboratory for Veterinary Diagnostics, D-59821 Arnsberg,4 Institute for Hygiene, University of Münster, D-48149 Münster, Germany,5 National Escherichia coli-Shigella Reference Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,2 Department of Biology, Yarmouk University, Irbid, Jordan3

Received 20 December 2001/ Returned for modification 4 February 2002/ Accepted 24 March 2002

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum 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, July 2002, p. 2555-2565, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2555-2565.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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