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2021 |
Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method |
Kevin Maafu Juma, Teisuke Takita, Kenji Ito, Masaya Yamagata,Shihomi Akagi, Emi Arikawa, Kenji Kojima, Manish Biyani,Shinsuke Fujiwara, Yukiko Nakura, Itaru Yanagihara, and Kiyoshi Yasukawa |
Biochemical and Biophysical Research Communications |
34166918 |
10.1016/j.bbrc.2021.06.023 |
Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method
Author(s):
Kevin Maafu Juma, Teisuke Takita, Kenji Ito, Masaya Yamagata,Shihomi Akagi, Emi Arikawa, Kenji Kojima, Manish Biyani,Shinsuke Fujiwara, Yukiko Nakura, Itaru Yanagihara, and Kiyoshi Yasukawa
Journal:
Biochemical and Biophysical Research Communications
Year:
2021
Abstract:
Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.
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