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2012 Fiscal Year Final Research Report

Development of discriminative and quantitative detection method for live and dead oral bacteria

Research Project

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Project/Area Number 22592341
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Social dentistry
Research InstitutionKyushu Dental College

Principal Investigator

YOSHIDA Akihiro  九州歯科大学, 歯学部, 助教 (20364151)

Co-Investigator(Kenkyū-buntansha) ANSAI Toshihiro  九州歯科大学, 歯学部, 准教授 (80244789)
SOH Inho  九州歯科大学, 歯学部, 助教 (10285463)
MINEOKA Tetsuro  九州歯科大学, 歯学部, その他 (50571402)
TAKEHARA Tadamichi  九州歯科大学, 歯学部, 教授 (00038879)
AWANO Shuji  九州歯科大学, 歯学部, 講師 (20301442)
Project Period (FY) 2010 – 2012
Keywords口腔細菌 / 生菌 / 死菌 / qPCR / 定量検出
Research Abstract

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important forunderstanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves 機関番号:27102研究種目: 基盤研究(C)研究期間: 平成22年度~平成24年度 課題番号:22592341研究課題名(和文) 生菌・死菌の区別が可能な新しい定量的口腔細菌検出法の開発研究課題名(英文) Development of discriminative and quantitative detection method for live and dead o … More ral bacteria研究代表者 吉田明弘(YOSHIDA, AKIHIRO)九州歯科大学・歯学部・助教研究者番号:20364151 for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. In addition, we applied this assay to analyze viable cell numbers in oral specimens. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens andthe oral status. Less

  • Research Products

    (26 results)

All 2013 2012 2011 2010 Other

All Journal Article (6 results) Presentation (14 results) Book (4 results) Remarks (1 results) Patent(Industrial Property Rights) (1 results) (of which Overseas: 1 results)

  • [Journal Article] Quantitative discrimination of Aggregatibacter actinomycetemicomitans highly leukotoxic JP2 clone form non-JP2 clones in diagnosis of aggressive periodontitis.2012

    • Author(s)
      Yoshida, A., Ennibi, O-K., Miyazaki, H., Hoshino, T., Hayashida, H., Nishihara, T., Awano, S. and Ansai, T
    • Journal Title

      BMC Infectious Dis.

      Volume: 12 Pages: 253

  • [Journal Article] 歯周病と細菌検査2012

    • Author(s)
      吉田明弘
    • Journal Title

      九州歯会誌

      Volume: 65 Pages: 169-178

  • [Journal Article] Streptococcus anginosusL-cysteine desulfhydrase gene expression is associated with abscess formation in BALB/c mice.2011

    • Author(s)
      Takahashi, Y., Yoshida, A., Nagata, E., Hoshino, T., Oho, T., Awano, S., Takehara, T. and Ansai, T.
    • Journal Title

      Molecular Oral Microbiol.

      Volume: 26 Pages: 221-227

  • [Journal Article] Enumeration of visible Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time Polymerase chain reaction.2011

    • Author(s)
      Takahashi, Y., Yoshida, A., Nagayoshi, M.,Kitamura, C., Nishihara, T., Awano, S.and Ansai, T
    • Journal Title

      Microbiol. Immunol.

      Volume: 55 Pages: 889-892

  • [Journal Article] The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization.2010

    • Author(s)
      Masakiyo, Y., Yoshida, A., Shintani, Y., Takahashi, Y., Ansai, T. and Takehara, T.
    • Journal Title

      Anaerobe

      Volume: 16 Pages: 265-269

  • [Journal Article] Rapid LED-based fluorescence microscopy distinguishes between live and dead bacteria in oral clinical samples.2010

    • Author(s)
      Masakiyo, Y., Yoshida, A., Takahashi, Y., Shintani, Y., Awano, S., Ansai, T., Sawayama, S., Shimakita, T. and Takehara, T
    • Journal Title

      Biomed Res.

      Volume: 31 Pages: 21-26

  • [Presentation] Proteome analysis of Streptococcus mutansbiofilms co-cultured with Streptococcus gordonii2012

    • Author(s)
      Yoshida, A., Yasunaga, A., Niki, M., Yamamoto, Y., Nishihara, T. and Ansai, T.
    • Organizer
      6th ASM Conference on Biofilms.
    • Place of Presentation
      Miami, Florida
    • Year and Date
      20120929-1004
  • [Presentation] Streptococcus gordoniiと共培養時にStreptococcus mutansに発現するタンパク質のプロテオーム解析2012

    • Author(s)
      吉田明弘、安永愛、仁木満美子、山本裕司、西原達次、中村卓、安細敏弘
    • Organizer
      第85回日本細菌学会総会
    • Place of Presentation
      長崎
    • Year and Date
      20120327-29
  • [Presentation] 生菌選択的なう蝕細菌定量法の開発と小児口腔内試料の解析.2012

    • Author(s)
      安永 愛、吉田明弘、正清義朗、森川和政、牧憲司、邵 仁浩、粟野秀慈、安細敏弘
    • Organizer
      第61回日本口腔衛生学会総会
    • Place of Presentation
      横須賀
    • Year and Date
      20120000
  • [Presentation] Aggregatibacter actinomycetemcomitansロイコトキシン産生/非産生株の選択的定量検出法の開発.2012

    • Author(s)
      吉田明弘、宮さき秀夫、林田秀明、粟野秀慈、安細敏弘
    • Organizer
      第61回日本口腔衛生学会総会
    • Place of Presentation
      横須賀
    • Year and Date
      20120000
  • [Presentation] 歯面初期定着菌群との共培養時におけるStreptococcus mutans抗酸化タンパク質の機能解析.2012

    • Author(s)
      安永 愛、吉田明弘、西原達次、安細敏弘
    • Organizer
      第54回日本歯科基礎医学会
    • Place of Presentation
      郡山
    • Year and Date
      20120000
  • [Presentation] Streptococcus gordoniiと共培養時にStreptococcus mutansに発現するタンパク質の解析.2011

    • Author(s)
      安永 愛、吉田明弘、仁木満美子、山本裕司、安細敏弘
    • Organizer
      日本細菌学会地方会
    • Place of Presentation
      門司
    • Year and Date
      20110800
  • [Presentation] 歯周病と細菌検査2011

    • Author(s)
      吉田明弘
    • Organizer
      九州歯科学会総会(北九州)シンポジウム
    • Place of Presentation
      北九州
    • Year and Date
      20110000
  • [Presentation] Enumeration of viable cariogenic pathogens assessed by PMA-based real-time PCR2011

    • Author(s)
      Yasunaga, A., Yoshida, A., Masakiyo, Y., Morikawa, K., Maki, K., Soh, I., Awano, S. and Ansai, T.
    • Organizer
      JADR
    • Place of Presentation
      広島
    • Year and Date
      20110000
  • [Presentation] Novel qualitative detection method for Aggregatibacter actinomycetemcomitans JP2.2010

    • Author(s)
      Dewake, N., Yoshida, A., Takahashi, Y., Awano, S., Miyazaki, H. and Ansai, T.
    • Organizer
      58th JADR
    • Year and Date
      20101120-21
  • [Presentation] Streptococcus mutansの生菌と死菌/損傷菌を区別した定量検出法の開発2010

    • Author(s)
      正清義朗、吉田明弘、高橋優介、安細敏弘
    • Organizer
      第59回日本口腔衛生学会・総会
    • Place of Presentation
      新潟
    • Year and Date
      20101007-08
  • [Presentation] Streptococcus anginosus βC-S lyase gene expression and experimental abscess formation.2010

    • Author(s)
      Yoshida, A., Takahashi, Y., Nagata, E., Hoshino, T., Oho, T., Awano, S. and Ansai, T.
    • Organizer
      第88回IADR
    • Place of Presentation
      Centre Convencions Internacional Barcelona
    • Year and Date
      20100714-17
  • [Presentation] 生菌・死菌を区別した根管内細菌の定量検出法の開発.2010

    • Author(s)
      高橋優介、吉田明弘、粟野秀慈、安細敏弘
    • Organizer
      第59回日本口腔衛生学会・総会
    • Place of Presentation
      新潟
    • Year and Date
      20100107-08
  • [Presentation] マウス実験膿瘍におけるStreptococcus anginosus lcd遺伝子の発現解析2010

    • Author(s)
      吉田明弘、高橋優介、長田恵美、於保孝彦、粟野秀慈、安細敏弘
    • Organizer
      第59回日本口腔衛生学会・総会
    • Place of Presentation
      新潟
    • Year and Date
      20100107-08
  • [Presentation] マウス膿瘍形成におけるStreptococcus anginosus βC-S liase遺伝子の発現解析.2010

    • Author(s)
      高橋優介、吉田明弘、新谷泰之、粟野秀慈、安細敏弘
    • Organizer
      第70回九州歯科学会総会
    • Place of Presentation
      北九州
    • Year and Date
      20100000
  • [Book] Oral disease and hydrogen sulfideproduction by oral bacteria (eds. Derin Adebayo and Aramide Okafor)2013

    • Author(s)
      Yoshida, A., Niki, M., Ansai, T. and Nakayama, K.
    • Total Pages
      131-145
    • Publisher
      Nova SciencePublishers, New York, USA
  • [Book] DHEAS and periodontal status in older Japanese. In: DHEA in Human Health and Aging, ed2012

    • Author(s)
      Yoshida, A. and Ansai, T.
    • Total Pages
      263-276
    • Publisher
      Ronald Ross Watson, CRC Press, Taylor & Francis Group LLC.
  • [Book] Microbiological Diagnosis for Periodontal Diseases. In: Periodontal Diseases - A Clinician's Guide (Ed. by Jane Manakil)2012

    • Author(s)
      Yoshida, A. and Ansai, T.
    • Total Pages
      55-66
  • [Book] 必修 臨床研修歯科医ハンドブック2010

    • Author(s)
      粟野秀慈、吉田明弘、邵 仁浩、安細敏弘
    • Publisher
      医歯薬出版
  • [Remarks]

    • URL

      http://www2.kyu-dent.ac.jp/dept/oral-health/

  • [Patent(Industrial Property Rights)] 口腔細菌の迅速検出2011

    • Inventor(s)
      吉村満美子、中山浩次、大原直也、吉田明弘、竹原直道
    • Industrial Property Rights Holder
      吉田明弘
    • Industrial Property Number
      特許第4792585号
    • Acquisition Date
      20110000
    • Overseas

URL: 

Published: 2014-08-29  

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