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

2019 Fiscal Year Final Research Report

• Project/Area Number: 16K06559
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Civil and environmental engineering
• Research Institution: Hachinohe 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
• 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

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.

Free Research Field

環境衛生工学

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。