期刊
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
卷 11, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fcimb.2021.632646
关键词
COVID-19; Crispr-Cas13a; saliva; SARS-CoV-2; CRISPR Diagnostics
资金
- UGC [F.4-5/2018 FRP]
- UGC-FRP startup grant [F.4.5 (236-FRP) 2015/BSR]
- DBT/Wellcome Trust India Alliance grant [IA/I/15/2/502086]
- SERB-ECR [ECR/2017/000976]
- Ramalingaswami Fellowship grant from Department of Biotechnology (DBT) [BT/RLF/Re-entry/09/2015]
- Early Career Research Award grant from Science and Engineering Research Board (SERB), Department of Science & Technology, Government of India [ECR/2018/002114]
- ICMR
A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and trained healthcare professionals, as well as constraints in the supply chain. CASSPIT introduces a new approach that allows direct use of saliva samples for detection without the extra RNA extraction step.
A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARSCoV-2 RNA spiked into the saliva samples was similar to 200 copies; image analysis-based quantification further improved the analytical sensitivity to similar to 100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.
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