| 研究開始時の研究の概要 |
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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| 研究実績の概要 |
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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