Solvent engineering studies on recombinase polymerase amplification

Kojima, Kenji; Juma, Kevin Maafu; Akagi, Shihomi; Hayashi, Kaichi; Takita, Teisuke; O’Sullivan, Ciara K.; Fujiwara, Shinsuke; Nakura, Yukiko; Yanagihara, Itaru; Yasukawa, Kiyoshi

doi:10.1016/j.jbiosc.2020.10.001

Cited by 22 publications

(21 citation statements)

References 25 publications

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“…We designed 13 factors and three concentrations (levels 1–3) for each factor ( Table S4 ). Level 2 was set as the concentration used in the standard condition with which we previously examined the effects of pH, CH 3 COOK concentration, and temperature on the RPA reaction efficiency [ 9 ]. Levels 1 and 3 were set as 25–50% and 200–400%, respectively, of level 2.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant uvsX, uvsY, and gp32 were expressed in Escherichia coli and purified from the cells as described previously [ 9 ]. The purified uvsX, uvsY, and gp32 preparations yielded a single band with molecular masses of 43, 22, and 34 kDa, respectively (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Such limitation makes the researchers have limited flexibility in exploring the effects of the concentration of each component on the reaction efficiency. In order to circumvent it, we previously prepared recombinant recombinase and SSB and used them to examine the effects of pH, temperature, and various additives on the efficiency of RPA [ 9 ]. In this study, we established the detection system of SARS-CoV-2 DNA and RNA and attempted to optimize the reaction condition, using one of the well-known statistical methods Taguchi method [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Juma

Takita

Ito

et al. 2021

Biochemical and Biophysical Research Communications

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Juma

Takita

Ito

et al. 2021

Biochemical and Biophysical Research Communications

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“…uvsX binds to the primers, while uvsY, originally identified as the T4 recombination mediator protein acts as the loading factor to assist uvsX to bind to the primers [ 7 ]. In earlier studies, we prepared recombinant uvsX, uvsY, and gp32 using an Escherichia coli expression system [ 8 ]. We also examined the effects of each component of the reaction solution on the RPA reaction efficiency and optimized the reaction conditions using a statistical method [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA

et al. 2022

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Background Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. Methods Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. Results The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 10 5 , 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. Conclusions The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA. Supplementary Information The online version of this article contains supplementary material available 10.1007/s11033-021-07098-y.

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“…As of now, we have not come across any study that has reported the co-assisted amplification of RNA and DNA cycles using an isothermal amplification method. In this study, we aimed to advance the bioengineering of our earlier studies on RNA-specific amplification 11 , 12 and RPA 8 , 9 , 21 and then integrate the essentials of both types of isothermal amplification methods into a simple format of ‘sample-in and answer-out’ with a primary focus on the detection of low copy numbers of viral RNA directly from COVID-19 saliva samples without the need for any laboratory handling or sample preprocessing. In this regard, we report the development of a completely homogeneous, isothermal, highly sensitive, and ultrarapid method for detecting virus RNA target sequences for the on-site (low resource settings) molecular diagnosis of COVID-19 and other infectious diseases.…”

Section: Introductionmentioning

confidence: 99%

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Biyani

Sharma

Kojima

et al. 2021

Sci Rep

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Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.

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Solvent engineering studies on recombinase polymerase amplification

Cited by 22 publications

References 25 publications

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

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