A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

• Main Authors: Iqbal Azmi, Md Imam Faizan, Rohit Kumar, Siddharth Raj Yadav, Nisha Chaudhary, Deepak Kumar Singh, Ruchika Butola, Aryan Ganotra, Gopal Datt Joshi, Gagan Deep Jhingan, Jawed Iqbal, Mohan C. Joshi, Tanveer Ahmad
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2021-03-01
• Series: Frontiers in Cellular and Infection Microbiology
• Subjects: COVID-19; Crispr-Cas13a; saliva; SARS-CoV-2; CRISPR Diagnostics
• Online Access: https://www.frontiersin.org/articles/10.3389/fcimb.2021.632646/full

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 SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity 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.