SARS‐CoV‐2 RNA Detection by a Cellphone‐Based Amplification‐Free System with CRISPR/CAS‐Dependent Enzymatic (CASCADE) Assay

Abstract

CRISPR (Clustered regularly interspaced short palindromic repeats)‐based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS‐CoV‐2 molecular detection at the point of need. However, efficient translation of CRISPR‐diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence‐based results readout. Herein, an amplification‐free CRISPR/Cas12a‐based diagnostic technology for SARS‐CoV‐2 RNA detection is presented using a smartphone camera for results readout. This method, termed C ellphone‐based a mplification‐free s ystem with C RISPR/C A S‐ d ependent e nzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase‐generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS‐CoV‐2 reverse‐transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL −1 ) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 – 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR‐positive COVID‐19 patients.

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…Additionally, a range of platforms have been developed by leveraging the advantages of CRISPR‐Cas with nucleic acid amplification technologies such as PCR (Ma, Peng, et al., 2021), RAA (Li, Ye, Chen, Xiang, et al., 2021), RPA (Chen et al., 2020; Zhuang et al., 2022), LAMP (Li, Chen, et al., 2022), multiple cross‐displacement amplification (Zhu et al., 2021), strand displacement amplification (Wang, Liu, et al., 2019; Zhou et al., 2018), rolling circle amplification (Qing et al., 2021), hybridization chain reaction (Liu et al., 2022), exponential amplification method (Huang et al., 2018; Tian et al., 2020), nicking enzyme‐assisted amplification (Bai et al., 2022), and so on. The CRISPR‐Cas biosensors are usually based on fluorescence signal readouts; other signal readouts such as colorimetry (Li, Zheng, et al., 2021), electrochemistry (Qing et al., 2021), lateral flow assay (Marsic et al., 2021), photothermal effect (Ma, Peng, et al., 2021), portable personal glucose meter (Liu, Hu, et al., 2021), surface‐enhanced Raman scattering (SERS) assay (Kim, Lee, Seo, et al., 2020; Pan et al., 2022), gas bubble signal (Silva et al., 2021), microfluidic paper‐based analytical device (μPAD) (Zhuang et al., 2022), hydrogel‐integrated paper‐based analytical device (μReaCH‐PAD) (Huang, Ni, et al., 2021), coupling biolayer interferometry (Qiao, Liu, et al., 2021), luminescence resonance energy transfer (Lin et al., 2022), and so on are established for visual and rapid biosensing applications.…”

The relevant information about the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the five-question approach (when, where, what, why, and how) and its impact on the environment

“…Furthermore, the Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) test is based on the Cas12-mediated transcleavage of a catalase: single-stranded DNA probe in response to the recognition of a specific nucleic acid target, such as SARS-CoV-2 genomic RNA. This generates a gas signal, which is subsequently identified using a smartphone-specific application and camera (Silva et al 2021). The use of CRISPR systems in molecular diagnostics and detection has developed, and Rahimi et al (2021) and Gupta et al (2021) have extensively described several CRISPRbased diagnostic methods for identifying COVID-19.…”

CRISPR-Powered Microfluidics in Diagnostics: A Review of Main Applications

“…In this regard, Silva et al developed a cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay (Figure 1A) [32]. After RNA extraction from nasal swabs, the prepared sample was combined with reverse transcriptase (RT) for about 10 min to produce RNA/DNA hybrids.…”

“…After the production of DNA amplicons, CRISPR/Cas12a specifically cleaved the target DNA so that the fluorescent signals could be observed. Integrating this into a digitized microfluidic chip, besides [32]. (B) deCOViD assay in which fluorophorequencher probes break after the activation of Cas12.…”