Development of novel high sensitive FISH for revealing the microbial ecology of uncultured microorganisms

• Project/Area Number: 24760437
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Civil and environmental engineering
• Research Institution: Anan National College of Technology
• Principal Investigator: KAWAKAMI Shuji 阿南工業高等専門学校, その他部局等, 助教 (00610461)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2012: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 高感度FISH法 / シングルセル解析 / 環境微生物学 / 環境微生物 / バイオリクター / FISH法 / in situ HCR法

Outline of Final Research Achievements

In this study, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR), and aptamer-based live-cell detection technique for revealing the microbial ecology of uncultured microorganisms. They are a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Research Products

• [Journal Article] In situ DNA-HCR: A facilitated in situ hybridization chain reaction system for the detection of environmental microorganisms. (2015)
  • Author(s): Tsuyoshi Yamaguchi, Shuji Kawakami, Masashi Hatamoto, Hiroyuki Imachi, Masanobu Takahashi, Nobuo Araki, Takashi Yamaguchi and Kengo Kubota.
  • Journal Title: Environmental microbiology Volume: 印刷中 Issue: 7 Pages: 2532-2541
  • DOI: 10.1111/1462-2920.12745
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] 高感度FISH法を用いた機能と系統を結びつけるシングルセル解析による脱窒素細菌の同定 (2013)
  • Author(s): 高橋竜司, 荒木信夫, 川上周司, 青木仁孝, 山口隆司.
  • Journal Title: 土木学会論文集G (環境) Volume: 68
  • NAID: 130004962579
  • Peer Reviewed
• [Journal Article] Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes. (2012)
  • Author(s): Kawakami, S., T. Hasegawa, Hiroyuki Imachi, Takashi Yamaguchi, Hideki Harada, Akiyoshi Ohashi, Kengo Kubota.
  • Journal Title: Journal of Microbiological Methods. Volume: 88 Pages: 218-223
  • Peer Reviewed
• [Presentation] 環境微生物の細胞壁タンパク質を検出するアプタマーの開発 (2015)
  • Author(s): 柿内涼太, 今出圭哉, 川上周司, 山田剛史, 山口剛士, 山口隆司.
  • Organizer: 第49回日本水環境学会年会
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-03-16 – 2015-03-18
• [Presentation] Modification of in situ DNA-HCR protocol for speedy detecting environmental microorganisms. (2015)
  • Author(s): 山口剛士, Bernhard M. Fuchs, 川上周司, 幡本将史, 久保田健吾, Rudolf Amann, 山口隆司.
  • Organizer: 第49回日本水環境学会年会
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-03-16 – 2015-03-18
• [Presentation] Applicability of in situ DNA-HCR for the detection of Archaea in anaerobic sludge (2014)
  • Author(s): Tsuyoshi YAMAGUCHI, Shuji Kawakami, Masashi HATAMOTO, Kengo KUBOTA, Nobuo ARAKI, Takashi YAMAGUCHI.
  • Organizer: 15th International Symposium on Microbial Ecology
  • Place of Presentation: Seoul, South-Korea
  • Year and Date: 2014-08-24 – 2014-08-29
• [Presentation] In situ HCR法を用いた活性汚泥中に生息する未培養微生物群の検出 (2014)
  • Author(s): 岡拓磨, 川上周司, 山口剛士, 山田剛史, 山口隆司
  • Organizer: 第48回日本水環境学会年会
  • Place of Presentation: 東北大学、仙台
• [Presentation] プタマーを用いた微生物の酵素の検出 (2014)
  • Author(s): 柿内涼太, 川上周司, 山口剛士, 山田剛史, 山口隆司
  • Organizer: 第48回日本水環境学会年会
  • Place of Presentation: 東北大学、仙台
• [Presentation] 酵素反応を用いない高感度in situ HCR法による環境微生物のmRNAの視覚的検出技術の開発 (2014)
  • Author(s): 大宮恭平, 山口剛士, 幡本将史, 川上周司, 久保田健吾, 中村明靖, 荒木信夫, 山口隆司
  • Organizer: 第48回日本水環境学会年会
  • Place of Presentation: 東北大学、仙台
• [Presentation] Detection of Archaea in an upflow anaerobic sludge blanket reactor by in situ hybridization chain reaction–fluorescence in situ hybridization (2013)
  • Author(s): Tsuyoshi YAMAGUCHI, Shuji Kawakami, Masashi HATAMOTO, Masanobu TAKAHASHI, Kengo KUBOTA, Hiroyuki IMACHI, Nobuo ARAKI, Takashi YAMAGUCHI
  • Organizer: The 13th World Congress on Anaerobic Digestion (AD13)
  • Place of Presentation: Santiago de compostela, Spain
• [Presentation] 新規蛍光増幅技術 (HCR) 法を用いた高感度FISH法の開発 (2013)
  • Author(s): 山口剛士, 川上周司, 幡本将史, 久保田健吾, 高橋優信, 井町寛之, 荒木信夫, 山口隆司
  • Organizer: 第16会日本水環境学会シンポジウム
  • Place of Presentation: 琉球大学、沖縄
• [Presentation] 豆腐工場排水を連続処理する酸素DHSリアクターの微生物相解析 (2013)
  • Author(s): 小島誠貴, 米澤愛美, 川上周司, 馬島孝治, 角野晴彦, 高石有希子, 珠坪一晃
  • Organizer: 第47回 日本水環境学会年会
  • Place of Presentation: 大阪工業大学
• [Presentation] TSA-FISH法を利用した脱窒素細菌のrRNAとnirK遺伝子の同時検出 (2013)
  • Author(s): 高橋竜司, 荒木信夫, 川上周司, 青木仁孝, 山口隆司
  • Organizer: 第47回 日本水環境学会年会
  • Place of Presentation: 大阪工業大学
• [Presentation] 酸素供給型スポンジ散水ろ床「Hi-DHSリアクター」による豆腐工場模擬排水の連続処理 (2013)
  • Author(s): 馬島孝治, 角野晴彦, 米澤愛美, 川上周司, 高石有希子, 珠坪一晃
  • Organizer: 第47回 日本水環境学会年会
  • Place of Presentation: 大阪工業大学
• [Presentation] A novel detection method for visualizing environmental microbes with low rRNA content and low cell permeability by using in situ HCR-FISH (2013)
  • Author(s): Tsuyoshi Yamaguchi, Shuji Kawakami, Masashi Hatamoto, Masanobu Takahashi, Kengo Kubota, Hiroyuki Imachi, Nobuo Araki, Takashi, Yamaguchi
  • Organizer: Annual Conference of the Association for General and Applied Microbiology (VAAM)
  • Place of Presentation: Congress Center Bremen, Germany
• [Presentation] Development of multi-staining in situ HCR for identification of prokaryotic. (2012)
  • Author(s): Hiroki Ohtsu, Shuji Kawakami, Hidemi Masuoka, Tsuyoshi Yamaguchi, Takashi Yamaguchi.
  • Organizer: The 28th Annual Meeting of the Japanese Society of Microbial Ecology (JSME2012)
  • Place of Presentation: Toyohashi University of Technology
• [Presentation] In situ HCR-FISH : a new sensitive fluorescence in situ hybridization technique for detecting environmental microorganisms (2012)
  • Author(s): Tsuyoshi Yamaguchi, Shuji Kawakami, Masashi Hatamoto, Masanobu Takahashi, Kengo Kubota, Hiroyuki Imachi, Nobuo Araki, Takashi, Yamaguchi
  • Organizer: 14 th International Symposium on Microbial Ecology (ISME14)
  • Place of Presentation: Copenhagen
• [Book] 生物燃料電池による廃水処理システム最前線 (2014)
  • Author(s): 山口隆司, 山口剛士, 幡本将史, 中村明靖, 川上周司, 久保田健吾
  • Total Pages: 14
  • Publisher: NTS出版