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

ABSTRACT

Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for assessing the infectivity of coronavirus disease 2019 and the efficacy of antiviral drugs. Here, we describe a digital warm-start CRISPR (WS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The WS-CRISPR assay combines low-temperature reverse transcription dual-priming mediated isothermal amplification (RT-DAMP) and CRISPR-Cas12a-based detection in one-pot, attributed to the mediation role by pyrophosphatase and phosphorothioated primers. The WS-CRISPR assay is initiated at above 50 {degrees}C and overcomes undesired premature target amplification at room temperature, enabling accurate digital nucleic acid quantification. By targeting SARS-CoV-2's nucleoprotein gene, digital WS-CRISPR assay is able to detect down to 5 copies/l SARS-CoV-2 RNA in the chip within 90 minutes. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples, showing 100% agreement with RT-PCR results. Moreover, the digital WS-CRISPR assay has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction, showing high tolerance to inhibitors. Thus, the digital WS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.