期刊
ACS NANO
卷 15, 期 1, 页码 1167-1178出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsnano.0c08165
关键词
RNA diagnostics; CRISPR-Cas13a; droplet microfluidics; liquid biopsy; SARS-CoV-2
类别
资金
- National Natural Science Foundation of China [21804044, 21475048, 21874049, 91959128]
- Guangzhou Science and Technology Program [201904010413]
- Fundamental Research Funds for the Central Universities [2019MS134]
- Guangdong Basic and Applied Basic Research Foundation [2020A1515010754]
The confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics eliminates the need for nucleic acid amplification and reverse transcription, achieving a >10,000-fold enhancement in sensitivity and enabling absolute digital single-molecule RNA quantitation. Its broad applicability and accurate detection of various RNA molecules at the single-molecule level make it a potential dominant rival to RT-qPCR.
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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