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.
Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for early diagnosis and timely medical treatment of coronavirus disease 2019. Here, we describe a digital warm-start CRISPR (dWS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The dWS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate and reliable digital quantification of SARS-CoV-2. By targeting SARS-CoV-2's nucleoprotein gene, the dWS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples. Moreover, it has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction. Thus, the dWS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.
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