1887

Abstract

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including , , and Shiga toxin-producing , 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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2004-07-01
2019-11-22
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References

  1. Asakura, H., Makino, S., Kobori, H., Watarai, M., Shirahata, T., Ikeda, T. & Takeshi, K. ( 2001;). Phylogenetic diversity and similarity of active sites of Shiga toxin (Stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals. Epidemiol Infect 127, 27–36.
    [Google Scholar]
  2. Cohen, N. D., Martin, L. J., Simpson, R. B., Wallis, D. E. & Neibergs, H. L. ( 1996;). Comparison of polymerase chain reaction and microbiological culture for detection of salmonellae in equine feces and environmental samples. Am J Vet Res 57, 780–786.
    [Google Scholar]
  3. Collins, E., Glennon, M., Hanley, S., Murray, A. M., Cormican, M., Smith, T. & Maher, M. ( 2001;). Evaluation of a PCR/DNA probe colorimetric membrane assay for identification of Campylobacter spp.in human stool specimens J Clin Microbiol 39, 4163–4165.[CrossRef]
    [Google Scholar]
  4. Doi, R., Ono, K., Saitoh, A., Ohtsuka, K., Shibata, Y. & Masaki, H. ( 2003;). Contamination levels of Salmonella and Listeria spp.in commercial raw meat. J Jpn Vet Med Assoc 56, 167–170.[CrossRef]
    [Google Scholar]
  5. Fukushima, H., Tsunimori, Y. & Seki, R. ( 2003;). Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J Clin Microbiol 41, 5134–5146.[CrossRef]
    [Google Scholar]
  6. Gentry-Weeks, C., Hutcheson, H. J., Kim, L. M., Bolte, D., Traub-Dargatz, J., Morley, P., Powers, B. & Jessen, M. ( 2002;). Identification of two phylogenetically related organisms from feces by PCR for detection of Salmonella spp. J Clin Microbiol 40, 1487–1492.[CrossRef]
    [Google Scholar]
  7. Infectious Disease Surveillance Center ( 2004;). Bacteria isolations from gastroenteritis. http://idsc.nih.go.jp
  8. Kehl, K. S., Havens, P., Behnke, C. E. & Acheson, D. W. K. ( 1997;). Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli. J Clin Microbiol 35, 2051–2054.
    [Google Scholar]
  9. Kulkarni, S. P., Lever, S., Logan, J. M., Lawson, A. J., Stanley, J. & Shafi, M. S. ( 2002;). Detection of campylobacter species: a comparison of culture and polymerase chain reaction based methods. J Clin Pathol 55, 749–753.[CrossRef]
    [Google Scholar]
  10. Lawson, A. J., Logan, J. M., O'Neill, G. L., Desai, M. & Stanley, J. ( 1999;). Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay. J Clin Microbiol 37, 3860–3864.
    [Google Scholar]
  11. Lin, J. S. & Tsen, H. Y. ( 1999;). Development and use of polymerase chain reaction for the specific detection of Salmonella typhimurium in stool and food samples. J Food Prot 62, 1103–1110.
    [Google Scholar]
  12. Linton, D., Lawson, A. J., Owen, R. J. & Stanley, J. ( 1997;). PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. J Clin Microbiol 35, 2568–2572.
    [Google Scholar]
  13. Logan, J. M. J., Edwards, K. J., Saunders, N. A. & Stanley, J. ( 2001;). Rapid identification of Campylobacter spp.by melting peak analysis of biprobes in real-time PCR. J Clin Microbiol 39, 2227–2232.[CrossRef]
    [Google Scholar]
  14. Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H. ( 1999;). Manual for Clinical Microbiology, 7th edn. Washington, DC: American Society for Microbiology.
  15. Ono, K., Tsuji, R., Ando, Y., Ohtsuka, K., Shibata, Y., Saitoh, A. & Masutani, T. ( 2003;). Contamination of Campylobacter spp.in domestic and imported chicken meat. J Jpn Vet Med Assoc 56, 103–105.[CrossRef]
    [Google Scholar]
  16. Ramotar, K., Waldhart, B., Church, D., Szumski, R. & Louie, T. J. ( 1995;). Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR. J Clin Microbiol 33, 519–524.
    [Google Scholar]
  17. Takeshi, K., Ikeda, T., Kubo, A. & 8 other authors ( 1997;). Direct detection by PCR of Escherichia coli O157 and enteropathogens in patients with bloody diarrhea. Microbiol Immunol 41, 819–822.[CrossRef]
    [Google Scholar]
  18. Wilson, I. G. ( 1997;). Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63, 3741–3751.
    [Google Scholar]
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