DataVXACS NanoPaper
journal article·2021-01-26

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

Tian Tian, Bowen Shu, Yongzhong Jiang, Miaomiao Ye, Lei Liu, Zhonghui Guo, Zeping Han, Zhang Wang, Xiaoming Zhou

ACS Nano· Volume 15· Issue 1
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Source abstract

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.
Source: openalex_inverted_index · Quality: baseline

Research context

Catalysis & surfaces

OpenAlex topics: Advanced biosensing and bioanalysis techniques · CRISPR and Genetic Engineering · Biosensors and Analytical Detection

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

Affiliations

School of Life Sciences, South China Normal University, Guangzhou 510631, China

Department of Laboratory Medicine, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou 510180, China

Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou 510180, China

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China

Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, Center for Studies of Psychological Application, South China Normal University, Guangzhou 510631, China

Department of Clinical Laboratory Medicine, Central Hospital of Panyu District, Guangzhou 511400, China

Institute of Ecological Science, School of Life Sciences, South China Normal University, Guangzhou 510180, China

Funding

Ministry of Education of the People's Republic of China · National Natural Science Foundation of China · Guangdong Basic and Applied Basic Research Foundation · Guangzhou Science and Technology Innovation Commission

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