Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis

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.

Considerations for diagnostic COVID-19 tests

“…Direct diagnostic testing to detect active SARS CoV2 infec tions mostly involves reverse transcriptase real time PCR (rtPCR), although different molecular techno logies, such as CRISPR mediated detection or loop mediated isothermal amplification, have also been appl ied [8][9][10][11][12] . Operation and application of these molecu lar tests is in keeping with those for previously developed tests that detect infectious agents 13 .…”

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers

“…Therefore, there is still an urgent need for more strategies that can be applied for rapid, accurate and sensitive diagnosis of COVID-19 patients even before an immune response can occur and more importantly for asymptomatic individuals. 14,15 Recent advances in structural biology have revealed that SARS-CoV-2 contains four main structural proteins, including the nucleocapsid (N), spike (S), envelope (E), and membrane (M) proteins. 16 Among them, the N protein is the most abundant, and relatively conserved protein in coronaviruses, which is involved in the transcription and replication of viral RNA.…”

One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a

“…Although RT-PCR can be used for quantitative analysis and OR-DETECTR cannot, the judgment of negative and positive results between the two platforms was consistent comparing CT and FC values (Figure 2a, 2b, S3, Table S2). 19,20). The amplification of viral nucleic acid is the key to whether it can be specifically detected.…”