Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

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 dWSCRISPR assay is initiated at above 50 degrees 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/mu l 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 SARSCoV-2 detection toward digitized quantification.

Automated summary

The study introduces a digital warm-start CRISPR assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples, overcoming undesired premature target amplification at room temperature. The method, targeting SARS-CoV-2's nucleoprotein gene, can detect down to 5 copies/ul SARS-CoV-2 RNA in the chip and has been clinically validated. Additionally, it can directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction, making it a sensitive and reliable CRISPR assay for accurate SARS-CoV-2 detection.