RESUMEN
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.
Asunto(s)
ADN Viral/análisis , ADN Polimerasa Dirigida por ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/normas , ARN Viral/análisis , Recombinasas/metabolismo , SARS-CoV-2/genética , Estadística como Asunto , Cartilla de ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , SARS-CoV-2/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
Recombinase polymerase amplification (RPA) is a technique that is used to specifically amplify a target nucleic acid sequence. Unlike the polymerase chain reaction (PCR), RPA is performed at a constant temperature between 37 and 42°C. Therefore, it can be potentially used for the onsite detection of various pathogens when combined with DNA extraction and amplicon detection techniques. In this study, we prepared recombinant recombinase and single-stranded DNA-binding protein from T4 phage and used them to examine the effects of reaction conditions and additives on the efficiency of RPA. The results revealed that the optimal pH was 7.5-8.0, optimal potassium acetate concentration was 40-80 mM, and optimal reaction temperature was 37-45°C although dimethyl sulfoxide at 5% v/v and formamide at 5% v/v inhibited the reaction. Our results suggest that RPA could be conducted using a wider range of optimal reaction conditions than those required for PCR and that RPA is highly suitable for point-of-care use.
