title: New immuno-PCR assay for detection of low concentrations of shiga toxin 2 and its variants.
abstract: 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.
authors.affiliation: Institute for Hygiene, University of Münster, Robert Koch Str. 41, 48149 Münster, Germany. tkuczius@uni-muenster.de.
authors.forename: Wenlan
authors.initials: W
authors.lastname: Zhang
authors.forename: Martina
authors.initials: M
authors.lastname: Bielaszewska
authors.forename: Matthias
authors.initials: M
authors.lastname: Pulz
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Friedrich
authors.forename: Helge
authors.initials: H
authors.lastname: Karch
authors.forename: Thorsten
authors.initials: T
authors.lastname: Kuczius
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000472:pathogenicity; D005243:Feces / Q000382:microbiology; D014644:Genetic Variation; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022641:Shiga Toxin 2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D022641:Shiga Toxin 2