Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

Abstract

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2’s nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

PfAgo-based detection of SARS-CoV-2

“…Even though RT-qPCR test is rapid, specific and economic, it takes hours to perform and requires specialized reagents, expensive equipment and skilled operators ( Li et al, 2019 ). To overcome these limitations, alternative nucleic acid detecting methods, especially programmable endonuclease based methods were innovated ( Ding et al, 2020 ) ( Guo et al, 2020 ) ( Hou et al, 2020 ) ( Joung et al, 2020 ) ( Lucia et al, 2020 ).…”

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA–CRISPR/Cas12a Assay

“…The RT-RPA pre-amplification step converts the RNA genome of the virus into dsDNA that serves as a substrate for the Cas12a cis activity. Cas12a targeting of the dsDNA triggers its collateral activity, which in turn cleaves the ssDNA reporter molecules and releasing the signal (Chen et al, 2018;Ding et al, 2020). We further combined the iSCAN-OP detection assay with a commercially available P51 fluorescence viewer device to facilitate quick, affordable, in-field diagnosis of plant RNA viruses.…”

Fluorescence polarization system for rapid COVID-19 diagnosis

“…The developed CODA platform distinguishes itself from other one-pot CRISPR/Cas assays ( Ding et al, 2020 ). By concurrently executing isothermal amplification and CRISPR/Cas detection in a single device, CODA eliminated extra hands-on steps, completing assays within a single sample loading workflow.…”