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

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.

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

“…Labeled amplification can be easily detected by using lateral flow strips ( Jiang et al., 2020a ; Lalremruata et al., 2020 ; Wu et al., 2020 ). The RPA system consists of three main proteins: recombinase, single-stranded DNA binding protein (SSB), and DNA polymerase ( Juma et al., 2021 ). Amplification is initiated by a primer-recombinase complex that invades the DNA double strand on the homologous sequence of the primer.…”

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

“…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 ]. In those studies, uvsX, uvsY, and gp32 were expressed as N- and C-terminal hexahistidine-tagged (His-tagged) proteins with a thrombin recognition site.…”

“…When treated with thrombin to cleave the His-tag, uvsY became insoluble while uvsX and gp32 remained soluble [ 8 ]. Therefore, we used untagged uvsX and gp32 and N- and C-terminal tagged uvsY to optimize reaction conditions [ 5 ]. However, it is possible that the uncleaved tag has a negative effect on the RPA reaction.…”

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

“…To obtain PCR-similar molecular testing outside a centralized laboratory, various isothermal (single-temperature) nucleic acid amplification methods have been devised and are in a continuous race to achieve a performance similar to that of PCR tests. The major candidates include recombinase polymerase amplification (RPA) 5 – 9 , nucleic acid sequence-based amplification (NASBA) or RNA-specific amplification 10 – 12 , and loop-mediated isothermal amplification (LAMP) 13 . The advantage of isothermal amplification methods over PCR is that they do not require bulky instrumentation, enable rapid (10–60 min) amplification of nucleic acids at a constant temperature (e.g., 37–65 °C) and therefore improve throughput in situations in which large numbers of clinical samples must be processed and facilitate point-of-care diagnosis.…”

“…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.…”