ABSTRACT
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.
Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , RNA Viruses/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Equipment Design , Humans , Limit of Detection , Nucleic Acid Amplification Techniques/instrumentation , RNA Viruses/isolation & purification , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Saliva/virologyABSTRACT
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.