Simultaneous detection of multiple aquatic microbes using TaqMan PCR system

2019 Fiscal Year Final Research Report

• Project/Area Number: 17K09268
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Legal medicine
• Research Institution: University of Miyazaki
• Principal Investigator: Yukawa Nobuhiro 宮崎大学, 医学部, 教授 (30240154)
• Co-Investigator(Kenkyū-buntansha): 柿崎 英二 宮崎大学, 医学部, 准教授 (70284833) ; 園田 愛 宮崎大学, 医学部, 助手 (10762122)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: 溺死の診断 / リアルタイムPCR / 水棲微生物 / 珪藻 / 水棲細菌

Outline of Final Research Achievements

We developed a new real-time PCR method to detect fresh and marine diatoms for diagnosis of drowning in various water environments. Present method was specific for various standard strains of fresh and marine diatoms (Achnantidium, Aulacoseira, Nitzschia, Tabellaria, Ulnaria, etc. as fresh water diatoms; Eucampia, Odontella, Pseudo-nitzschia, Thalassionema, etc. as marine diatoms). The sensitivity of the method was >10 pg/tube. Moreover, the diatom DNAs were detectable in water samples collected from various water environments although they were undetectable in the blood and tissues (kidney and liver) from cadavers retrieved from the rivers and sea by the new method. These results gave us one of the reasons to prove our hypothesis that the diatom DNAs would be almost undetectable in blood and closed organs (kidney and liver).

Free Research Field

法医学

Academic Significance and Societal Importance of the Research Achievements

海水域や淡水域など生息水域に限らず，一つのプライマーセットを用いて一括して珪藻類を検出可能な測定系の開発に取り組んだ。開発した測定系は珪藻以外の他の水棲微生物との交叉性（偽陽性）を示さず，検出限界は1～10 pg/tubと良好であった。ところで従来の珪藻検査から得られる結果の意義，特に肺以外の諸臓器や血液から得られる結果の意義については，世界中で意見が分かれ未だ解決には至っていない。本研究によって得られた成果はこの問題に対しいくつかの重要な示唆を与えることができたと考えている。今後も引き続き検討を重ねこの課題の解決に繋げたい。