Abstract
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.
Highlights
The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world
Severe acute respiratory syndrome Coronavirus 2 (SARSCoV-2, previously named 2019-nCoV) is a new coronavirus causing coronavirus disease 2019 (COVID-19) which first emerged in December 20191
Polymerase chain reaction (PCR) method including its variant reverse transcription PCR (RT-PCR) is the most commonly used technology for pathogen nucleic acid detection and has been considered as a gold standard for infectious disease diagnostics due to its high sensitivity and specificity[3,4,5]. It typically relies on expensive equipment and well-trained personnel, as well as needs long assay reaction time (~2 h), all of which is not suitable for simple, rapid, and point of care (POC) molecular diagnostics of the SARS-CoV-2
Summary
The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Polymerase chain reaction (PCR) method including its variant reverse transcription PCR (RT-PCR) is the most commonly used technology for pathogen nucleic acid detection and has been considered as a gold standard for infectious disease diagnostics due to its high sensitivity and specificity[3,4,5]. It typically relies on expensive equipment and well-trained personnel, as well as needs long assay reaction time (~2 h), all of which is not suitable for simple, rapid, and point of care (POC) molecular diagnostics of the SARS-CoV-2. Our AIOD-CRISPR method has been validated by testing 28 clinical swab samples and obtained consistent results with that of RT-
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