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Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay
Xiong Ding; Kun Yin; Ziyue Li; Maroun M. Sfeir; Changchun Liu.
Affiliation
  • Xiong Ding; University of Connecticut Health Center
  • Kun Yin; University of Connecticut Health Center
  • Ziyue Li; University of Connecticut Health Center
  • Maroun M. Sfeir; University of Connecticut Health Center
  • Changchun Liu; University of Connecticut Health Center
Preprint in En | PREPRINT-MEDRXIV | ID: ppmedrxiv-20236109
Journal article
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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-2s 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.
License
cc_by_nc_nd
Full text: 1 Collection: 09-preprints Database: PREPRINT-MEDRXIV Type of study: Diagnostic_studies / Prognostic_studies Language: En Year: 2020 Document type: Preprint
Full text: 1 Collection: 09-preprints Database: PREPRINT-MEDRXIV Type of study: Diagnostic_studies / Prognostic_studies Language: En Year: 2020 Document type: Preprint
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