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Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay

¹Department of Microbiology, Kobe Institute of Health, Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan ²Department of Microbiology, Tenri Hospital, Mishima-cho, Tenri 632-8552, Japan

Correspondence Yoshio Iijima iijima{at}oak.ocn.ne.jp

Received January 21, 2004
Accepted March 26, 2004

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40 %) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70 %) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18 %) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6 %) patients were found to be positive by PCR analysis.

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