Abstract
Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.
Keywords: COVID-19; CRISPR; Fluorescent detection; Highly sensitive; Molecular diagnosis; SARS-CoV-2.
【저자키워드】 COVID-19, SARS-CoV-2, CRISPR, Molecular diagnosis, Highly sensitive, Fluorescent detection, 【초록키워드】 SARS-CoV-2, equipment, diagnostic, RT-PCR, diagnostics, amplification, RT-qPCR, Asymptomatic, Real-time PCR, qPCR, pre-symptomatic, Research, limit of detection, nasal swab, Quantitative, analytical sensitivity, RPA, Amplicon, isothermal, criteria, COVID-19 case, individual, complex, average, fluorescent, positive, polymerase, probe, infect, Inconclusive, lab, approach, recent, highlight, robust, IMPROVE, greater, produced, performed, detect, reported, assays, required, facilitate, exhibited, demonstrated, comparable, SARS-CoV-2-infected individual, 【제목키워드】 COVID-19 diagnosis,