Multiplex Solid-Phase RPA Coupled CRISPR-Based Visual Detection of SARS-CoV-2

Abstract

AbstractCOVID-19 has challenged the world’s public health and led to over 4.5 million deaths. A rapid, sensitive, and cost-effective point-of-care virus detection device is crucial to the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Here we demonstrate a solid phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive and visual COVID-19 DNA detection. By targeting the SARS-CoV-2 structure protein encoded genomes, two specific genes were simultaneously detected with the control sample without cross-interaction with other sequences. The endpoint signal can be directly visualized for rapid detection of COVID-19. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter released fluorescent signal when specific genes were recognized. The system was tested with samples of a broad range of concentrations (20 to 2×105 copies) and showed analytical sensitivity down to 20 copies per reaction. Furthermore, a low-cost LED UV flashlight (∼$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method with the capacity to specifically identify low concentration genes. Similar CRISPR biosensor chips can support a broad range of applications such as HPV DNA detection, influenza SARS-CoV-2 multiplex detection, and other infectious disease testing assays.HighlightsA commercially available UV flashlight excited CRISPR-Cas12a based visual sensor combined with solid-phase RPAThe developed assay could detect SARS-CoV-2 down to 20 copies with no signal crosswalkThis multiplexed on-chip virus detection system would provide great insight for detecting and differentiating SARS-CoV-2 from other viral infections with a single sample simultaneously.