Development of Digital Isothermal Nucleic Acid Amplification for Rapid Quantification

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K15334
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory medicine
• Research Institution: University of Yamanashi (2018); Institute of Physical and Chemical Research (2016-2017)
• Principal Investigator: Tanaka Yuji 山梨大学, 大学院総合研究部, 特任准教授 (40625513)
• Co-Investigator(Kenkyū-buntansha): 寺尾 泰久 順天堂大学, 医学部, 先任准教授 (00348997)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: 等温核酸増幅 / デジタル / 逆転写 / ゲノム変異 / 融解曲線解析 / 再生医療 / 網膜再生

Outline of Final Research Achievements

In this study, we examined two new detection methods: digital nucleic acid quantification method with reverse transcription and digital nucleic acid quantification method of identifying and quantifying nucleic acid amplified by melting curve analysis. The isothermal amplification method can amplify a nucleic acid only by maintaining it at around 70 ° C, but there is a problem that it can not be determined unless there is a 10-fold concentration difference, but digitization of the isothermal amplification kit with reverse transcription It has been found that it becomes possible to detect even a concentration difference of about 2 times. In addition, in order to detect genomic mutations used for quality control of cells utilized for regenerative medicine of retina, newly designed primers for isothermal amplification, digital nucleic acid amplification, and biotyped and mutated nucleic acid by melting curve analysis. It turned out that it can be identified and quantified.

Free Research Field

トランスレショナルリサーチ

Academic Significance and Societal Importance of the Research Achievements

ゲノムをあらかじめ調べた上でその患者に適した薬剤を処方する「個別化医療」の実用化が始まり、より簡易的に、より正確に、より迅速に、より低価格で、より多様な核酸の差異を識別するための技術開発の重要性が高まっている。本研究でRNAの迅速簡易定量とゲノム変異の融解曲線解析による定量という２つの新たな検出方法の可能性を見出した。既存の核酸検出の改良や、新たな個別化医療や再生医療の創出に貢献する可能性がある。