Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples |
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Authors: | Wiemer Dorothea Loderstaedt Ulrike von Wulffen Hinrik Priesnitz Simone Fischer Marcellus Tannich Egbert Hagen Ralf Matthias |
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Affiliation: | aDepartment for Tropical Medicine at the Bernhard Nocht Institute, Bundeswehr Hospital Hamburg, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany;bInstitute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany;cMedilys Institute for Laboratory Medicine, Microbiology and Hygiene, Paul-Ehrlich-Strasse 1, 22763 Hamburg, Germany;dBernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany |
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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 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 103 cfu mL−1 for Campylobacter jejuni, 104 cfu mL−1 for Salmonella, and 105 cfu mL−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. |
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Keywords: | Real-time PCR Multiplex PCR Salmonella Shigella Yersinia Campylobacter jejuni |
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