An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.
ACS Nano
; 15(1): 1167-1178, 2021 01 26.
Article
in English
| MEDLINE | ID: covidwho-1014984
ABSTRACT
Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10â¯000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA
/
Microfluidics
/
CRISPR-Cas Systems
Type of study:
Diagnostic study
/
Prognostic study
/
Randomized controlled trials
Limits:
Humans
Language:
English
Journal:
ACS Nano
Year:
2021
Document Type:
Article
Affiliation country:
Acsnano.0c08165
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