A Recombinase Polymerase Amplification-LFA Paper Chip for the Naked-Eye Detection of SARS-CoV-2

Abstract

The COVID-19 pandemic has exhibited high transmissibility even in asymptomatic individuals, resulting in significant global consequences. The COVID-19 has emphasized the critical need for rapid and accurate detection, particularly during the early stages of infectious disease. The World Health Organization (WHO)-recommended real-time polymerase chain reaction (RT-PCR) provides high sensitivity but requires specialized equipment, trained technician, and consumes a long time for processing, which limit its practicality for widespread use. In contrast, rapid antigen tests offer ease of use but are limited by relatively low sensitivity. To overcome these limitations, this study proposes a novel diagnostic platform that combines the high sensitivity of RT-PCR with the speed and convenience of lateral flow immunoassay (LFA). In this study, we developed point-of-care testing (POCT) device based on isothermal amplification for the detection of SARS-CoV-2. The platform combines recombinase polymerase amplification (RPA), an isothermal amplification method that eliminates the need for thermal cycling equipment, with lateral flow immunoassay (LFA) to enable visual detection of amplification results. The entire diagnostic process, including amplification (~20 minutes) and detection (~10 minutes), is completed within 30 minutes. Additionally, the platform is designed with a single sample inlet for simple and user-friendly operation, making it highly suitable for POCT applications. The platform successfully detected purified SARS-CoV-2 RNA at concentrations as low as 10 copies. Amplification and detection processes were fully integrated into a single platform, and recombinase polymerase amplification (RPA) amplicons visually detected using LFA through simple temperature modulation. This platform as the potential for sensitive and rapid detection of not only SARS-CoV-2 RNA but also a wide range of target RNAs and DNAs, emphasizing its flexibility and suitability as a robust tool for POCT.