Development of effective pre-treatment method for the detection of Mycobacteria

Research Project

Project/Area Number	18K12125
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Review Section	Basic Section 90130:Medical systems-related
Research Institution	公益財団法人結核予防会結核研究所
Principal Investigator	Mitarai Satoshi 公益財団法人結核予防会結核研究所, 抗酸菌部, 部長 (30501671)
Project Period (FY)	2018-04-01 – 2021-03-31
Project Status	Completed (Fiscal Year 2020)
Budget Amount *help	¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000) Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000) Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords	結核菌 / 検体処理 / 抗酸菌濃縮 / 誘電泳動 / 核酸増幅法 / 診断 / 高感度化 / 検体濃縮
Outline of Final Research Achievements	An effective concentration method is required to detect Mycobacterium tuberculosis (MTB) in paucibacillary specimens. Dielectrophoresis (DEP), a phenomenon in which a force is exerted on a dielectric particle subjected to a non-uniform electric field, is useful for concentrating bacteria. To investigate whether the DEP method increases nucleic acid amplification test (NAAT) sensitivity. First, the capture rates were examined for multiple electrode settings by calculating the bacterial load before and after DEP with real-time PCR. Second, LAMP was performed using samples with 10 consecutive negative results by conventional LAMP and DEP treated samples. The capture rate was the highest with 100 kHz frequency (73.2-84.9%). LAMP with DEP was performed using conventional LAMP-negative specimens, and eight of ten tests (80%) were positive. The sensitivity was higher than that of the conventional LAMP method (p = 0.0007). The DEP method has the potential to increase NAAT sensitivity for MTB.
Academic Significance and Societal Importance of the Research Achievements	誘電泳動法によって抗酸菌（結核菌）の効率的濃縮条件を決定した。またその技術により結核菌の核酸増幅法による検出感度を上昇させることが可能であることを実証した。これは現状の結核菌核酸増幅法検査の弱点である検体濃縮過程に寄与する技術であり、学術的には抗酸菌の誘電泳動濃縮が可能であることを証明し、社会的にはこの技術の応用で結核菌検査の高感度化・迅速診断に寄与しうる。