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An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.

An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.

Authors :
Tian T
Shu B
Jiang Y
Ye M
Liu L
Guo Z
Han Z
Wang Z
Zhou X
Source :
ACS nano [ACS Nano] 2021 Jan 26; Vol. 15 (1), pp. 1167-1178. Date of Electronic Publication: 2020 Dec 17.
Publication Year :
2021

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 >10 000-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.

Details

Language :
English
ISSN :
1936-086X
Volume :
15
Issue :
1
Database :
MEDLINE
Journal :
ACS nano
Publication Type :
Academic Journal
Accession number :
33498106
Full Text :
https://doi.org/10.1021/acsnano.0c08165