Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/140518
Title: Developing a rapid, specific and sensitive CRISPR/Cas12a-based test kit for in-vitro diagnosis of COVID-19
Authors: Teo, Seok Yee
Keywords: Science::Biological sciences::Molecular biology
Science::Biological sciences::Genetics
Issue Date: 2020
Publisher: Nanyang Technological University
Abstract: SARS-CoV-2 is the causative agent of the ongoing COVID-19 pandemic. The outbreak originated in Wuhan, China, and has since spread throughout the world. Given the severity of disease and high transmission potential of SARS-CoV-2, rapid, specific and sensitive assays are required to meet diagnostic needs related to the outbreak. A recent report has described the use of a Cas12a-based method to detect SARS-CoV-2 with high specificity and sensitivity. However, the workflow is time-consuming and require multiple heating sources, making it incompatible for field application. Here, we improve the field usability of Cas12a detection by using a fluorescence-based screening assay to identify a Cas12a protein, enAsCas12a, that can specifically detect SARS-CoV-2 at room temperature. Using mismatched gRNA, we found that enAsCas12a can tolerate single-nucleotide mismatches throughout the target sequence, making it robust against viral evolution. In addition, RT-LAMP-Cas12a detection conditions were optimized to reduce overall sample-to-result time by 8 min without compromising sensitivity. Although results are inconclusive, we demonstrate that Cas12a detection may be visualized on a lateral flow strip for easy result interpretation. Finally, different gRNA targeting SARS-CoV-2 were screened that show higher signals than gRNA previously reported for SARS-CoV-2 detection.
URI: https://hdl.handle.net/10356/140518
Fulltext Permission: restricted
Fulltext Availability: With Fulltext
Appears in Collections:SBS Student Reports (FYP/IA/PA/PI)

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