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New Immuno-PCR Assay for Detection of Low Concentrations of Shiga Toxin 2 and Its Variants

Institute of Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Robert Koch Str. 41, 48149 Münster, Germany,¹ Governmental Institute of Public Health of the Lower Saxony, Roesebeckstr. 4-6, 30449 Hannover, Germany,² Institute of Medical Microbiology, University of Münster, Domagkstr. 10, 48149 Münster, Germany³

Received 23 November 2007/ Returned for modification 2 January 2008/ Accepted 1 February 2008

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

Published ahead of print on 13 February 2008.