Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19)

Abstract

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19) Jean-Marc Zingg a,*, Yu-Ping Yang a, Spencer Seely a, Pratibha Joshi a, Md Harun Or Roshid a,b, Fabiola Iribarren Latasa a,c, Gregory O'Connor a, Jennifer Alfaro d, Eduardo Riquelme d, Sebastian Bernales d,e, Emre Dikici a, f, Sapna Deo a, f,**, Sylvia Daunert a, f,g,*** a Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL, 33136-6129, USA b Department of Chemistry, University of Miami, Miami, FL, 33146, USA c Universidad Francisco de Vitoria, Madrid, Spain d Merken Biotech SpA, Za~nartu, 1482, Santiago, Chile e Centro Ciencia & Vida, Za~nartu, 1482, Santiago, Chile f Dr. JT Macdonald Foundation Biomedical Nanotechnology Institute, University of Miami, Miami, FL, 33136-6129, USA g University of Miami Clinical and Translational Science Institute, University of Miami, Miami, FL, 33136-6129, USA A R T I C L E I N F O Handling Editor: Prof Angelo M. Azzi Keywords: Coronavirus Point of care (PoC) test Lateral flow assay (LFA) Recombinase polymerase amplification (RPA) Isothermal amplification Reverse transcription recombinase polymerase amplification (RT-RPA) A B S T R A C T Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrumentbased whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in presymptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P < 0.05, CI: 78.2–93.8%) and specificity of 93.9% (P < 0.05, CI: 85.4–97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors’ offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.