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 RNA extraction 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 94 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. 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.

Clinical validation of RCSMS: a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva

“…The resulting molecular test, RCSMS, can be easily implemented at the primary care level and diagnostic laboratories alike, using a simple lateral flow readout. To our knowledge, this is the first clinical validation of a CRISPR-Cas-based test for COVID-19 in Latin America using saliva, and one of the few of its type developed worldwide (33, 34, 35). Noteworthy, an Argentinian group recently reported a DETECTR system (36) that uses nasopharyngeal swabs and fluorescence readouts.…”

“…The resulting molecular test, RCSMS, can be easily implemented at the primary care level and diagnostic laboratories alike, using a simple lateral flow readout. To our knowledge, this is the first clinical validation of a CRISPR-Cas-based test for COVID-19 in Latin America using saliva, and one of the few of its type developed worldwide (33,34,35). Edgardo Rebagliati National Hospitals, for their generous and selfless participation in this study, despite their precarious condition and the often pressing circumstances; for their words of encouragement towards Peruvian science, and for their great humanity.…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…This kit was designed to detect ORF 1ab of SARS-CoV-2 and its superior sensitivity and specificity have made it a good candidate to be employed in rapid diagnosis of SARS-CoV-2 [175] . Next, an Indian company introduced an extraction-free test kit that was based on the CRISPR/Cas-based detection technology and this test assay was aimed to detect S and ORF 1ab of SARS-CoV-2 [176] . This test kit was reported to have a similar LOD as the ViroReal ® Kit RT-LAMP SARS-CoV-2 kit (100 RNA copies in a reaction) and the sensitivity and specificity of this CRISPR/Cas-based test kit was reported to be almost 100% [176] .…”

“…Next, an Indian company introduced an extraction-free test kit that was based on the CRISPR/Cas-based detection technology and this test assay was aimed to detect S and ORF 1ab of SARS-CoV-2 [176] . This test kit was reported to have a similar LOD as the ViroReal ® Kit RT-LAMP SARS-CoV-2 kit (100 RNA copies in a reaction) and the sensitivity and specificity of this CRISPR/Cas-based test kit was reported to be almost 100% [176] . On the other side, an extraction-free assay that was dependent on the nucleic acid sequencing technology was introduced and approved for use to detect SARS-CoV-2 and it was reported to be able to detect 3200 RNA copies in 1 mL [177] .…”

“…However, the genetic targets that are targeted by this rapid diagnostic assay and its specificity were unclear [177] . By comparing the test sensitivity and specificity of the extraction-free diagnostic assays that are based on RT-LAMP, CRISPR/Cas, and nucleic acid sequencing [175] , [176] , [177] , it is found that these assays have comparable good sensitivity and specificity to detect SARS-CoV-2 by targeting various molecular targets of the virus. Thus, more focus should also be given to extraction-free diagnostic tests that are based on the non-RT-PCR approach to increase the selectability and options for the users to choose and use.…”