key: cord-0870746-ssdtw255
authors: Juma, Kevin Maafu; Takita, Teisuke; Ito, Kenji; Yamagata, Masaya; Akagi, Shihomi; Arikawa, Emi; Kojima, Kenji; Biyani, Manish; Fujiwara, Shinsuke; Nakura, Yukiko; Yanagihara, Itaru; Yasukawa, Kiyoshi
title: Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method
date: 2021-06-10
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.06.023
sha: 7549de92435c26a3d1cbe1a3e5e7c01daa732203
doc_id: 870746
cord_uid: ssdtw255

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.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a 27 target DNA sequence with a recombinase, a single-stranded DNA-binding protein 28 (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the 29 reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical 30 method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA 31 were used as targets. After evaluating the concentration of each component, the uvsY, 32 gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the 33 balance between the binding and dissociation of uvsX and DNA primer was precisely Buckinghamshire, UK). The purified DNA and RNA concentrations were determined 79 spectrophotometrically at A 260 . The DNA and RNA were stored at -30ºC or -80ºC, 80 respectively.

In System 1, the standard DNA was amplified by PCR using 400nt_F and 400nt_B 82 as a pair of primers and the mixture of oligonucleotides 400nt-1, 400nt-2, and 400nt-3 83 as a template. The T7 promoter-bearing standard DNA was amplified by PCR using 84 T7-400nt_F and 400nt_B as a pair of primers and the standard DNA as a template 85 (Table S1 ). Standard RNA was synthesized by in vitro transcription using the T7 86 promoter-bearing standard DNA as a template. In System 2, the standard DNA was 87 amplified by PCR using 300nt_F and 300nt_B as a pair of primes and the mixture of 88 oligonucleotides 300nt-1 and 300nt-2 as a template. The T7 promoter-bearing standard 89 DNA was amplified by PCR using T7-300nt_F and 300nt_B as a pair of primers and the 90 J o u r n a l P r e -p r o o f standard DNA as a template (Table S2) . Standard RNA was synthesized by in vitro 91 transcription using the T7 promoter-bearing standard DNA as a template. The reaction mixture (30 L) for RPA was designed and prepared according to 106 Taguchi's L27 (Table S3) 128

phosphoprotein gene was selected as a target according to the previous report [13] . We 139 designed three forward and three reverse primers (Table S1) respectively, of level 2. According to Taguchi's L27 orthogonal array (Table S3) Thirteen factors and each three levels in Round 3 were determined (Table S6) . (Table S2 ) and selected one combination (2F-15 204 and 2R-11) that exhibited the best performance in sensitivity. The size of amplified 205 product by the primer combination was 99 bp.

RPA was carried out with 60−6 ×10 7 copies of standard DNA, and RT-RPA was 207 carried out with 60−6 ×10 7 copies of standard RNA, both at 41°C for 1 h. In the 208 analysis of the RPA or RT-RPA products in the subsequent electrophoresis, the 209 optimized conditions detected 60 copies of standard DNA ( Fig. 2A) or RNA (Fig. 2B) .

Finally, we compared the sensitivities of RPA before and after optimization. Using

System 2, RPA was carried out with 60−6 ×10 7 copies of standard DNA (Fig. S3) . The 212 condition after optimization detected 60 copies of standard DNA while that before 213 optimization did not detect 600 copies. These results indicated that by optimizing the 214 reaction conditions for three enzymes, 100 to 1000-fold higher sensitivity was achieved. 

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