期刊
ANALYTICA CHIMICA ACTA
卷 1205, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.aca.2022.339749
关键词
CRISPR/Cas13; Point-of-Care; SARS-CoV-2; Diagnostics; Gold nanoparticles; Naked-eye
资金
- Spanish Ministry of Economy and Competitiveness [SAF2017-87305-R, PID2020-119352RB-I00]
- Instituto de Salud Carlos III [FONDO-COVID19:COV20/00144, COV20/00122]
- Madrid Regional Government [PEJD-2017-PRE/BMD-3730, PEJD-2018-PRE/IND-9584, PEJD-2017-PRE/IND-4438, IND2017/IND7809]
- Ministry of Economy, Industry and competitiveness of Spain [BES-2017.082521]
- 'Severo Ochoa' Programme for Centres of Excellence in RD (MINECO) [SEV-2016-0686, CEX2020-001039-S]
- Madrid Regional Government (NANOCOV-CM)
The COVID-19 pandemic highlights the necessity of fast and sensitive detection methods to prevent pathogen spread. In this study, the authors present CASCADE, a sensing system that allows rapid naked-eye detection of SARS-CoV-2 RNA. CASCADE utilizes LwaCas13a CRISPR protein and gold nanoparticles to recognize and visualize target RNA.
The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases. (C) 2022 The Authors. Published by Elsevier B.V.
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