CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.
Sens Actuators B Chem
; 362: 131765, 2022 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-35370361
ABSTRACT
SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.
COVID-19; COVID-19, coronavirus disease 2019; CRISPR, clustered regularly interspaced short palindromic repeats; CRISPR-Cas13; Cas, CRISPR associated proteins; LOD, limit of detection; NASBA, nucleic acid sequence-based amplification; Nucleic acid tests; RNA aptamer; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; T4 PNK, T4 Polynucleotide Kinase; crRNA, CRISPR RNA
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Sens Actuators B Chem
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
China