A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/μL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.
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| http://dx.doi.org/10.1016/j.bios.2022.114393 | DOI Listing |
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