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Development of an automated, DNA amplification technique-based method for detection of food-poisoning bacteria

Research Project

Project/Area Number 04557024
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 細菌学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

NISHIBUCHI Mitsuaki  Kyoto Univ., Fac.of Medicine Associate Professor, 医学部, 助教授 (50189304)

Co-Investigator(Kenkyū-buntansha) FUKUSHIMA Shigeru  Shimazu Corporation, Central Research Laboratory, Corporate R/D Operations Princ, 技術研究本部中央研究所, 主任研究員
KURAZONO Hisao  Kyoto Univ., Fac.of Medicine, Assistant Professor, 医学部, 助手 (90186487)
TAKEDA Yoshifumi  Kyoto Univ., Fac.of Medicine, Professor, 医学部, 教授 (30029772)
高野 純  島津製作所, 技術研究本部中央研究所, 主任研究員
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 1993: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1992: ¥9,600,000 (Direct Cost: ¥9,600,000)
KeywordsFood-poisoning bacteria / Vibrio parahaemolyticus / Enterotoxigemic Escherichia coli / Vero toxin / Cholera toxin / PCR method / DNA amplification method / Vero毒素 / 病原大腸菌 / 黄色ブドウ球菌 / エンテロトキシン遺伝子
Research Abstract

The purpose of this research project was to develop a quick and easy system to confirm food poisoning cases by combining the DNA amplification method or polymerase chain reaction (PCR) method for a particular target gene of the causative bacterium and an automated sample-processing and DNA-detecting equipment. The most important point was to establish an approach to obtain the oligonucleotide primers and amplification conditions which are the key factors determining the specificity and sensitivity of detection. We have adapted an empirical but sure method ; we repeatedly screened many test strains to determine the two parameters for each target gene of the food-poisoning bacterium. We then obtained primer sets and amplification conditions for the following target genes : the thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes of Vibrio parahaemolyticus, heat-labile and -stable enterotoxin genes and Vero toxin genes of Escherichia coli, enterotoxin genes and toxic shock syndrome toxin genes of Staphylococcus aureus, and cholera toxin gene of Vibrio cholerae.
We also developed a method to detect amplified DNA fragments so that the above PCR can be performed in an automated equipment. The PCR products were labeled with biotin, trapped through affinity with streptoavidin onto the wells of a 96-well microtiter plate. The trapped PCR products were detected by oligonucleotide probes labeled nonisotopically (alkaline phosphatase). The feature of our detection method was that a few copies of the trget gene could be detected in 45 minites which is faster than any other microtiter-based non-isotopic methods reported so far.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Tada,J.: "Detection of the thermostable direct hemolysin gene(tdh)and the thermostable direct hemolysin-related hemolysin gene(trh)of Vibrio parahaemolyticus." Mol.Cell.Probes. 6. 477-487 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tada,J.: "Nonisotopic microtiter plate-based assay for detecting products of polymerase chain reaction amplification:application to detection of the tdh gene of Vibrio parahaemolyticus" Mol.Cell.Probes. 6. 489-494 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kishishita,M.: "Sequence Variation in the thermostable direct hemolysin-related hemolysin(trh)gene of Vibrio parahaemolyticus" Appl.Environ.Microbiol.58. 2449-2457 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 西渕光昭: "PCRによる腸炎ビブリオの耐熱性溶血毒遺伝子および類似毒素遺伝子の検出法" 感染症(日本臨床特別号). 50. 348-352 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 西渕光昭: "PCR法を用いた下痢原性細菌の検出" 医学のあゆみ. 163. 172 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tada, T.et al.: "Detection of the thermostable direct hemdysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus" Mol.Cell.Probes. 6. 447-487 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tada, T.et al.: "Nonisotopic microtiter plate-based assay for detecting products of polymerase chain reaction amplification : application to detection on the tdh gene of Vibrio parahaemolyticus" Mol.Cell.Probes. 6. 489-494 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kishishita, M.et al.: "Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus" Appl.Environ.Microbiol.58. 2449-2457 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nishibuchi, M.et al.: "Methods to detect the thermostable direct hemolysin gene and a related hemolysin gene of Vibrio parahaemolyticus by PCR (in Japanese)" Nippon rinsho. 50. 348-352 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nishibuchi, M.et al.: "Detection of diarrheagenic bacteria by the PCR method (in Japanese)" J.Clin.Expt.Med.(IGAKU NO AYUMI). 163. 172

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tada,J.: "Detection of the thermostable direct hemolysin gene(tdh)and the thermostable direct hemolysin-related hemolysin gene(trh)of Vibrio parahaemolyticus by polymerase chain reaction" Mol.Cell.Probes. 6. 477-487 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Tada,J.: "Nonisotopic microtiter plate-based assay for detecting products of polymerase chain reaction amplification:application to dection of the tdh gene of Vibrio parahaemolyticus" Mol.Cell.Probes. 6. 489-494 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kishishita,M.: "Sequence variation in the thermostable direct hemolysin-related hemolysin(trh)gene of Vibrio parahaemolyticus" Appl.Environ.Microbiol.58. 2449-2457 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] 西渕光昭: "PCRによる腸炎ビブリオの耐熱性溶血毒遺伝子および類似遺伝子の検出法" 感染症(日本臨床特別号). 50. 348-352 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] 西渕光昭: "PCR法を用いた下痢原性細菌の検出" 医学のあゆみ. 163. 172 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Tada,J.: "Detection of the thermostable direct hemolysin gene(tdh)and the thermostable direct hemolysin-related hemolysin gene(trh)of Vibrio Parahaemolyticus by polymerase chain reaction" Mol.Cell.Probes. 6. 477-487 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Tada,J.: "Nonisotopic microtiler plate-based assay for detecting products of polymerase chain reaction amplification:application to detection of the tdh gene of Vibrio parahaemolyticus" Mol.Cell.Probes. 6. 489-494 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kishishita,M.: "Sequence variation in the thermostable direct hemolysin-related hemolysin(trh)gene of Vibrio parahaemolyticus" Appl.Environ.Microbiol.58. 2449-2457 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 西渕 光昭: "PCRによる腸炎ビブリオの耐熱性溶血毒遺伝子および類似毒素遺伝子の検出法" 感染症(日本臨床特別号). 348-352 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 西渕 光昭: "PCR法を用いた下痢原性細菌の検出" 医学のあゆみ. 163. 172- (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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