Optimization of Reaction Condition of Recombinase Polymerase Amplification to Detect SARS-CoV-2 DNA and RNA Using a Statistical Method

Overview

Journal Biochem Biophys Res Commun

Publisher Elsevier

Specialty Biochemistry

Date 2021 Jun 24

PMID 34166918

Citations 4

Authors

Kevin Maafu Juma

Teisuke Takita

Kenji Ito

Masaya Yamagata

Shihomi Akagi

Emi Arikawa

Kenji Kojima

Manish Biyani

Shinsuke Fujiwara

Yukiko Nakura

Itaru Yanagihara

Kiyoshi Yasukawa

Affiliations

Soon will be listed here.

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.

Citing Articles

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

Morimoto K, Juma K, Yamagata M, Takita T, Kojima K, Suzuki K Mol Biol Rep. 2024; 51(1):367.

PMID: 38411701 PMC: 10899321. DOI: 10.1007/s11033-024-09367-y.

Preliminary Evaluation of Rapid Visual Identification of Using a Newly Developed Lateral Flow Strip-Based Recombinase Polymerase Amplification (LF-RPA) System.

Li J, Zhong Q, Shang M, Li M, Jiang Y, Zou J Front Cell Infect Microbiol. 2022; 11:804737.

PMID: 35118011 PMC: 8804217. DOI: 10.3389/fcimb.2021.804737.

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

Juma K, Takita T, Yamagata M, Ishitani M, Hayashi K, Kojima K Mol Biol Rep. 2022; 49(4):2847-2856.

PMID: 35098395 PMC: 8801280. DOI: 10.1007/s11033-021-07098-y.

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

Biyani R, Sharma K, Kojima K, Biyani M, Sharma V, Kumawat T Sci Rep. 2021; 11(1):15997.

PMID: 34362977 PMC: 8346491. DOI: 10.1038/s41598-021-95411-x.

References

Hashimoto K, Yonesaki T . The characterization of a complex of three bacteriophage T4 recombination proteins, uvsX protein, uvsY protein, and gene 32 protein, on single-stranded DNA. J Biol Chem. 1991; 266(8):4883-8. View

Hidese R, Kawato K, Nakura Y, Fujiwara A, Yasukawa K, Yanagihara I . Thermostable DNA helicase improves the sensitivity of digital PCR. Biochem Biophys Res Commun. 2017; 495(3):2189-2194. DOI: 10.1016/j.bbrc.2017.12.053. View

Corman V, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu D . Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3). PMC: 6988269. DOI: 10.2807/1560-7917.ES.2020.25.3.2000045. View

Al-Madhagi S, Joda H, Jauset-Rubio M, Ortiz M, Katakis I, OSullivan C . Isothermal amplification using modified primers for rapid electrochemical analysis of coeliac disease associated DQB1*02 HLA allele. Anal Biochem. 2018; 556:16-22. DOI: 10.1016/j.ab.2018.06.013. View

Luo G, Yi T, Jiang B, Guo X, Zhang G . Betaine-assisted recombinase polymerase assay with enhanced specificity. Anal Biochem. 2019; 575:36-39. DOI: 10.1016/j.ab.2019.03.018. View

Thanakiatkrai P, Welch L . Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I. Int J Legal Med. 2011; 126(1):161-5. DOI: 10.1007/s00414-011-0558-5. View

Roperch J, Benzekri K, Mansour H, Incitti R . Improved amplification efficiency on stool samples by addition of spermidine and its use for non-invasive detection of colorectal cancer. BMC Biotechnol. 2015; 15:41. PMC: 4446959. DOI: 10.1186/s12896-015-0148-6. View

Sabate Del Rio J, Steylaerts T, Henry O, Bienstman P, Stakenborg T, Van Roy W . Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification. Biosens Bioelectron. 2015; 73:130-137. DOI: 10.1016/j.bios.2015.05.063. View

Piepenburg O, Williams C, Stemple D, Armes N . DNA detection using recombination proteins. PLoS Biol. 2006; 4(7):e204. PMC: 1475771. DOI: 10.1371/journal.pbio.0040204. View

10.

Jauset-Rubio M, Tomaso H, El-Shahawi M, Bashammakh A, Al-Youbi A, OSullivan C . Duplex Lateral Flow Assay for the Simultaneous Detection of Yersinia pestis and Francisella tularensis. Anal Chem. 2018; 90(21):12745-12751. DOI: 10.1021/acs.analchem.8b03105. View

11.

Kojima K, Juma K, Akagi S, Hayashi K, Takita T, OSullivan C . Solvent engineering studies on recombinase polymerase amplification. J Biosci Bioeng. 2020; 131(2):219-224. DOI: 10.1016/j.jbiosc.2020.10.001. View

12.

Lobato I, OSullivan C . Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt Chem. 2020; 98:19-35. PMC: 7112910. DOI: 10.1016/j.trac.2017.10.015. View

13.

Yasukawa K, Mizuno M, Konishi A, Inouye K . Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis. J Biotechnol. 2010; 150(3):299-306. DOI: 10.1016/j.jbiotec.2010.09.961. View

14.

Li J, Macdonald J, von Stetten F . Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst. 2018; 144(1):31-67. DOI: 10.1039/c8an01621f. View

15.

Ishino Y . Studies on DNA-related enzymes to elucidate molecular mechanisms underlying genetic information processing and their application in genetic engineering. Biosci Biotechnol Biochem. 2020; 84(9):1749-1766. DOI: 10.1080/09168451.2020.1778441. View

16.

Yasukawa K, Yanagihara I, Fujiwara S . Alteration of enzymes and their application to nucleic acid amplification (Review). Int J Mol Med. 2020; 46(5):1633-1643. PMC: 7521554. DOI: 10.3892/ijmm.2020.4726. View

17.

Okano H, Katano Y, Baba M, Fujiwara A, Hidese R, Fujiwara S . Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase. Enzyme Microb Technol. 2016; 96:111-120. DOI: 10.1016/j.enzmictec.2016.10.003. View

18.

Spiess A, Mueller N, Ivell R . Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem. 2004; 50(7):1256-9. DOI: 10.1373/clinchem.2004.031336. View

19.

Gajewski S, Waddell M, Vaithiyalingam S, Nourse A, Li Z, Woetzel N . Structure and mechanism of the phage T4 recombination mediator protein UvsY. Proc Natl Acad Sci U S A. 2016; 113(12):3275-80. PMC: 4812704. DOI: 10.1073/pnas.1519154113. View

20.

Kikuchi A, Sawamura T, Kawase N, Kitajima Y, Yoshida T, Daimaru O . Utility of spermidine in PCR amplification of stool samples. Biochem Genet. 2010; 48(5-6):428-32. DOI: 10.1007/s10528-009-9326-3. View