title: Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples.
abstract: Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10(3)cfumL(-1) for Campylobacter jejuni, 10(4)cfumL(-1) for Salmonella, and 10(5)cfumL(-1) for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples (n=192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.
authors.affiliation: Department for Tropical Medicine at the Bernhard Nocht Institute, Bundeswehr Hospital Hamburg, Germany. Wiemer@bni-hamburg.de
authors.forename: Dorothea
authors.initials: D
authors.lastname: Wiemer
authors.forename: Ulrike
authors.initials: U
authors.lastname: Loderstaedt
authors.forename: Hinrik
authors.initials: H
authors.lastname: von Wulffen
authors.forename: Simone
authors.initials: S
authors.lastname: Priesnitz
authors.forename: Marcellus
authors.initials: M
authors.lastname: Fischer
authors.forename: Egbert
authors.initials: E
authors.lastname: Tannich
authors.forename: Ralf Matthias
authors.initials: RM
authors.lastname: Hagen
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D003967:Diarrhea / Q000382:microbiology*; D004755:Enterobacteriaceae / Q000235:genetics / Q000302:isolation & purification*; D004756:Enterobacteriaceae Infections / Q000175:diagnosis* / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors