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

Abstract

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.

Lateral flow assays (LFA) as an alternative medical diagnosis method for detection of virus species: The intertwine of nanotechnology with sensing strategies

“…The intensity of the TL was interpreted using Fiji image J software to quantitatively detect amplified RNAs. The device showed a 98% agreement with RT-qPCR results [ 81 ].…”

Paper-Based Point-of-Care Testing of SARS-CoV-2

“…This assay also achieved excellent sensitivity and specificity. However, a LOD of 100 copies/μl could potentially impact its utility ( Azmi et al, 2021 ).…”

High‐Affinity Dimeric Aptamers Enable the Rapid Electrochemical Detection of Wild‐Type and B.1.1.7 SARS‐CoV‐2 in Unprocessed Saliva

“…There are several examples of both nucleic acid and antigen tests (typically nucleocapsid (N)‐protein or spike (S)‐protein), including lateral flow assays for genomic RNA[ 12 , 13 , 14 , 15 , 16 ] and S‐protein, [17] as well as electrochemical sensors for detection of genomic RNA, [18] N‐protein [19] or S‐protein. [ 20 , 21 , 22 ] However, at present these rapid saliva tests either require complicated and time‐consuming separation, [22] RNA extraction[ 15 , 16 ] or amplification steps,[ 13 , 18 , 23 ] have not been validated with clinical samples, [21] require long assay times (>1 h),[ 12 , 13 , 14 , 15 , 16 ] or do not provide adequate detection limits (<1000 viral copies per mL,[ 11 , 17 , 19 , 20 , 21 , 22 , 24 , 25 , 26 , 27 , 28 ] corresponding to a RT‐PCR cycle threshold ( C t ) of 36). [29] Given these issues, there remains a need for simple, rapid and sensitive SARS‐CoV‐2 tests, which should be possible to achieve via detection of viral surface proteins (i.e., spike proteins) directly in unprocessed saliva.…”