Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2

被引:69
作者
Ali, Zahir [1 ]
Sanchez, Edith [1 ]
Tehseen, Muhammad [2 ]
Mahas, Ahmed [1 ]
Marsic, Tin [1 ]
Aman, Rashid [1 ]
Rao, Gundra Sivakrishna [1 ]
Alhamlan, Fatimah Saeed [3 ]
Alsanea, Madain S. [3 ]
Al-Qahtani, Ahmed A. [3 ]
Hamdan, Samir [2 ]
Mahfouz, Magdy [1 ]
机构
[1] 4700 King Abdullah Univ Sci & Technol, Div Biol Sci, Lab Genome Engn & Synthet Biol, Thuwal 239556900, Saudi Arabia
[2] King Abdullah Univ Sci & Technol KAUST, Biol & Environm Sci & Engn Div, Lab DNA Replicat & Recombinat, Thuwal 239556900, Saudi Arabia
[3] King Faisal Specialist Hosp & Res Ctr, Dept Infect & Immun, Riyadh 11211, Saudi Arabia
关键词
COVID-19; SARS-CoV-2; variants; lateral flow assay; CRISPR-dCas9 and dCas9; nucleic acid detection; biotin labelling; IN-VIVO; PRINCIPLES; BIOTINYLATION; PROTEINS;
D O I
10.1021/acssynbio.1c00499
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/mu L) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the alpha, beta, and delta SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.
引用
收藏
页码:406 / 419
页数:14
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