Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection

Abstract

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5′-6FAM 3′-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

Detect and destroy: CRISPR-based technologies for the response against viruses

“…Similarly, a SHERLOCK assay was validated on >150 patient samples in Thailand having strong concordance with RT-qPCR ( Patchsung et al., 2020 ). Many more publications soon followed ( Ackerman et al., 2020 ; Ali et al., 2020 ; Arizti-Sanz et al., 2020 ; Chen et al., 2020 ; Fozouni et al., 2021 ; Guo et al., 2020 ; Hou et al., 2020 ; Huang et al., 2020 ; Joung et al., 2020 ; Ning et al., 2020 ; Ramachandran et al., 2020 ; Wang et al., 2020b ) with methods summarized in Table 1 .…”

“…There are two approaches to combining amplification and detection reactions in a single tube: (1) physical separation of the reaction components within the same tube that can be mixed later or (2) identification of reaction conditions that eliminate the need for amplification or are compatible for both amplification and CRISPR-based cleavage. The former approach still requires the user to manipulate the tube, albeit without opening it, and has been tested using mineral oil ( Chen et al., 2020 ) or placing reagents on the side of the tube ( Ali et al., 2020 ) or in the tube’s cap ( Guo et al., 2020 ), followed by shaking the tube after the initial incubation. Thermophilic Cas12 orthologs were used in HOLMESv2 and STOPCovid so that RT-LAMP and Cas12 detection could be performed in a single tube.…”

“…Both Cas13- and Cas12-based diagnostics have methods that capture fluorescent collateral cleavage via a portable commercial or a 3D-printed, blue-light-emitting device. In order to streamline analysis, smartphone applications have been developed and used to interpret and report results ( Arizti-Sanz et al., 2020 ; Chen et al., 2020 ; Fozouni et al., 2021 ; Ning et al., 2020 ). Furthermore, for low-titer samples, the signal of the lateral flow test band is often difficult to distinguish from the background signal.…”

Trends in biosensing platforms for SARS-CoV-2 detection: A critical appraisal against standard detection tools

“…The test displayed no cross reactivity to other coronaviruses in the detection of SARS-CoV-2 viral RNAs. Chen’s group introduced contamination free naked-eye detection of SARS-CoV-2 using RT-LAMP approach as shown in Figure 1 B [ 56 ]. The portable 3D printing instrument and smartphone were used and the created fluorescence was seen by naked eyes without using some devoted tools.…”

“…The S1 subunit of spike protein consist of N-terminal domain (NTD), receptor binding domain (RBD) and C-terminal domain while the S2 subunit comprises heptad repeat 1and 2, which plays a critical role in viral entry, (B) The flow chart diagram describing the whole experimental process and principle for contamination-free visual detection method to detect different concentrations of RNA. Reprinted with permission [ 56 ]. …”