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Evaluation of PMA-PCR and RT-PCR methods combined with 16S rRNA illumina sequencing techniques for detection of live bacterial community

Research Project

Project/Area Number 16K06559
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Civil and environmental engineering
Research InstitutionHachinohe National College of Technology

Principal Investigator

YAGUCHI JUNICHI  八戸工業高等専門学校, その他部局等, 教授 (80342450)

Co-Investigator(Kenkyū-buntansha) 山本 歩  八戸工業高等専門学校, その他部局等, 准教授 (60523800)
Project Period (FY) 2016-04-01 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2019: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2017: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2016: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywordsアンプリコンシーケンス解析 / PMA / rRNA / 病原性細菌 / RT-PCR / β多様性解析 / 次世代シーケンサー / PCR / ろ過濃縮 / 細菌叢 / RNA / rRNA / 大腸菌 / 環境 / 土木環境システム / 細菌 / 感染症 / バイオテクノロジー
Outline of Final Research Achievements

In this study, PCR with selective membrane-permeable dye, PMA(Propidium monoazide) (PMA-PCR) and RNA targeted reverse transcription PCR(RT-PCR) combined with illumina 16S rRNA sequencing techniques were developed to detect viable microbial community in environmental water samples. Microbial community analysis of the heated-treated primary effluent spiked with two viable enteric bacteria showed that the amplicon sequencing based on PMA-PCR and RT-PCR methods were effective for the detection of physiologically active bacterial community. Especially, RT-PCR method based on RNA was able to detect both of active enteric bacteria more appropriately than PMA-based methods. Four environmental samples including primary effluent, chlorinated effluent, river water and digested sludge were assessed the viability of bacterial community using PCR, PMA-PCR and RT-PCR method. Three methods had their quite different community structures from each other according to beta diversity analysis.

Academic Significance and Societal Importance of the Research Achievements

本研究は、大腸菌など指標生物に基づく糞便汚染の評価法を革新するスタートの研究と位置付けることができ、研究開発した生理的活性のある細菌群のみを菌叢解析する手法によって、活性を維持している病原性細菌の水環境中での挙動を把握することができ、感染リスクの評価と汚染源対策を通じてリスクマネジメントに大きく貢献できる。開発した手法を2つの下水処理場に適用して病原性細菌の挙動解析を行ったところ、Mycobacterium属の細菌が塩素消毒後も活性を維持して残存していることが分かった。

Report

(5 results)
  • 2019 Annual Research Report   Final Research Report ( PDF )
  • 2018 Research-status Report
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (8 results)

All 2020 2019 2018

All Journal Article (1 results) (of which Peer Reviewed: 1 results) Presentation (7 results) (of which Int'l Joint Research: 1 results)

  • [Journal Article] 表紙2019

    • Author(s)
      日脇陸生、矢口淳一
    • Journal Title

      RESEACH REPORTS National Institute of Technology,Hachinohe College

      Volume: 53 Issue: 0 Pages: Cover1

    • DOI

      10.24704/hnctech.53.0_Cover1

    • NAID

      130007633982

    • ISSN
      0385-4124, 2433-2003
    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Presentation] 下水処理プロセスにおける生理的活性のある細菌群の挙動解析2020

    • Author(s)
      矢口淳一  成田健志 Nur Dianna binti Ibraham
    • Organizer
      令和元年度土木学会東北支部技術研究発表会
    • Related Report
      2019 Annual Research Report
  • [Presentation] 排水処理プロセスにおける活性のある病原性細菌の挙動について2020

    • Author(s)
      矢口淳一  成田健志 Nur Dianna binti Ibraham
    • Organizer
      第54回日本水環境学会年会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Enumeration of Viable Escherichia coli in Wastewater and River Water Utilizing RNA-Based RT-qPCR Method2019

    • Author(s)
      Junichi Yaguchi, Narita Kenshi, Satomi Nagamori
    • Organizer
      The 8th IWA-ASPIRE Conference & Exhibition 2019
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 効果的な生理的活性のある細菌の網羅的解析2019

    • Author(s)
      日脇陸生、山本 歩、矢口淳一
    • Organizer
      第53回日本水環境学会年会
    • Related Report
      2018 Research-status Report
  • [Presentation] PMAxx試薬による生理的活性のある細菌の分別効果2019

    • Author(s)
      成田健志、高島 慧周、矢口淳一
    • Organizer
      平成30年度土木学会東北支部技術研究発表会
    • Related Report
      2018 Research-status Report
  • [Presentation] 次世代シーケンサーを用いた生理的活性のある細菌の網羅的解析2018

    • Author(s)
      日脇陸生、山本 歩、矢口淳一
    • Organizer
      第55回環境フォーラム(土木学会)
    • Related Report
      2018 Research-status Report
  • [Presentation] rRNAを利用した生存可能な大腸菌の計数について2018

    • Author(s)
      長森 郷仁, 成田 健志, 矢口 淳一
    • Organizer
      第52回日本水環境学会年会
    • Related Report
      2017 Research-status Report

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Published: 2016-04-21   Modified: 2021-02-19  

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