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An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics
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.
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Metadata
- Authors
- Tian, T., Shu, B., Jiang, Y., Ye, M., Liu, L., Guo, Z., Han, Z., Wang, Z., & Zhou, X.
- Venue
- ACS Nano
- Publisher
- American Chemical Society (ACS)
- Published
- 2020-12-17
- Volume
- 15
- Issue
- 1
- Pages
- 1167-1178
- ISSN
- 1936-0851 (ISSN-L)
- 1936-086X
- OpenAlex
- W3111292947
- Cited by
- 328
- References
- 43
- FWCI
- 11.89
- Type
- journal article
- Language
- en
- Open access
- closed
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