Abstract
Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.
Highlights
The outbreak of the novel coronavirus SARS-CoV-2, the causative agent of the COVID19 pandemic, poses a significant threat to all aspects of human life (Wu et al, 2020)
We report an efficient, isothermal, one-step/one-pot reverse transcription-recombinase polymerase amplification (RT-RPA) method coupled with the Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system for the simple, specific, rapid, and sensitive detection of SARS-CoV-2 RNA in clinical samples
Among the different primer sets screened for Cas12a and Cas12b in the twoiSCAN-V2 for POC SARS-CoV2 Detection pot assay, only a single set of primers worked in the one-pot assay with both proteins (Supplementary Figures S1A,B)
Summary
The outbreak of the novel coronavirus SARS-CoV-2, the causative agent of the COVID19 pandemic, poses a significant threat to all aspects of human life (Wu et al, 2020). Preventive measures have been in place to minimize the number of new cases, mass screening is essential to identify and isolate SARS-CoV-2-infected individuals to limit virus spread and alleviate the burden on healthcare systems (Khan et al, 2020; Mercer and Salit, 2021). Additional implementation of effective measures, including testing, tracking, and tracing, is vital to limit transmission and control the spread of the virus (Güner et al, 2020). Nucleic acid-based diagnostic systems are the most valued and effective methods for identifying a virus in a particular sample type. Reverse transcription-quantitative PCR (RTqPCR) remains the gold standard for detecting SARS-CoV-2 in patient samples, the need for well-equipped laboratories, expensive reagents, and trained personnel hinders its use in resource-limited areas. There is a pressing need for portable and easy-to-deploy diagnostics at the point-of-care (POC) (Morshed et al, 2007; Sharma et al, 2015; Corman et al, 2020; Rezaei et al, 2020)
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