title: Evaluation of 5'-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools.
abstract: 5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with reference strains of E. coli (n=40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx(1c), stx(1d), and stx(2f)), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log(10) and 3.42 to 4.63 log(10) CFU/g of stool for 5'-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation of </=5% for both inter- and intra-assay variation. Clinical performance was identical with a panel of archived positive specimens (n=19) and a prospective panel of stools associated with bloody diarrhea (n=115). In conclusion, both assays proved to be sensitive and reproducible.
authors.affiliation: Department of Research and Development, Laboratory for Infectious Diseases, Groningen, The Netherlands.
authors.forename: Tim
authors.initials: T
authors.lastname: Schuurman
authors.forename: Alexander
authors.initials: A
authors.lastname: Roovers
authors.forename: W Kim
authors.initials: WK
authors.lastname: van der Zwaluw
authors.forename: Anton A
authors.initials: AA
authors.lastname: van Zwet
authors.forename: Luc J M
authors.initials: LJ
authors.lastname: Sabbe
authors.forename: A Mirjam D
authors.initials: AM
authors.lastname: Kooistra-Smid
authors.forename: Yvonne T H P
authors.initials: YT
authors.lastname: van Duynhoven
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis; D000818:Animals; D002522:Chlorocebus aethiops; D015342:DNA Probes / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000032:analysis; D005243:Feces / Q000382:microbiology; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics; D022603:Shiga Toxins / Q000096:biosynthesis* / Q000235:genetics; D019914:Taq Polymerase / Q000737:chemistry / Q000378:metabolism; D014709:Vero Cells
chemical_list: D018829:Adhesins, Bacterial; D015342:DNA Probes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C439650:SAA protein, E coli; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D022603:Shiga Toxins; D019914:Taq Polymerase