Abstract
The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay’s specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.
Keywords: COVID-19; CRISPR; CRISPR-Top; Cas12b; Nucleic acid detection; SARS-CoV-2.
【저자키워드】 COVID-19, SARS-CoV-2., CRISPR, Nucleic acid detection, CRISPR-Top, Cas12b, 【초록키워드】 coronavirus disease, SARS-CoV-2, Coronavirus disease 2019, coronavirus, primary care, diagnostic test, diagnostic, risk, severe acute respiratory syndrome Coronavirus, RT-PCR, Laboratory, RNA, cross-reactivity, lateral flow, CRISPR, amplification, clinical samples, nucleic acid, DNA, SARS-CoV-2 detection, specificity, Contamination, Nucleic acid detection, diagnostic tests, limit of detection, target, temperature, ORF1ab, followed by, causative agent, Non-COVID-19, acute respiratory syndrome, Frame, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, reaction, These data, positive result, complexity, clinic, swab samples, positive, cross-contamination, clinical sample, performed, detect, collected, clinically, applied, can be used, increase, 【제목키워드】 application, platform, diagnosis of COVID-19,