| Project/Area Number |
23KJ1201
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund |
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| Section | 国内 |
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| Review Section |
Basic Section 38030:Applied biochemistry-related
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| Research Institution | Kyoto University |
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Principal Investigator |
Juma Kevin 京都大学, 農学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2023-04-25 – 2024-03-31
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| Project Status |
Discontinued (Fiscal Year 2023)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2024: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2023: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | DNA amplification / Thermostable enzyme / Lyophilization / Pyruvate kinase |
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| Outline of Research at the Start |
In this study, recombinant enzymes used in RPA for detection of SARS-CoV-2 DNA and RNA and other pathogens will be prepared. Glycerol will be removed from the RPA enzyme solutions and lyophilized. Optimization of lyophilized reagents including trehalose concentrations will be performed. Storage conditions and sensitivity of lyophilized reagents will be evaluated. Finally, a field survey will be carried out in Kenya.
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| Outline of Annual Research Achievements |
My research focused on establishing an isothermal recombinase polymerase amplification (RPA), a method conducted at around 41°C, utilizing recombinase, single-stranded DNA binding protein, strand-displacing DNA polymerase, and an ATP regenerating enzyme. I engineered a pyruvate kinase (Tma-PK) from thermophilic bacterium Thermotoga maritima as the ATP regenerating enzyme in RPA. I successfully optimized lyophilization conditions for RPA. The lyophilized RPA reagents remained stable for over 28 days at room temperature. RPA reagents containing Tma-PK exhibited superior performance to those with human PK suggesting that thermostable enzymes are preferable to mesophilic ones in RPA. I disseminated these findings in two domestic conferences and published two peer-reviewed articles.
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