A sensitive electrochemical method for rapid detection of dengue virus by CRISPR/Cas13a-assisted catalytic hairpin assembly.

Dengue fever caused by Dengue virus (DENV) infection has been widely popular, especially in tropical and subtropical areas. Rapid and sensitive diagnosis is the first priority for treatment of DENV infection. This work designed a signal amplification strategy for sensitive electrochemical detection of DENV by using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system for catalytic hairpin assembly on electrode surface. The presence of target RNA could activate the cleavage activity of the CRISPR/Cas13a system to release the blocker silenced swing arms, which then hybridized with hairpin 1 (H1) immobilized on electrode surface to expose the pre-locked toehold domain of H1 for the hybridization of ferrocene-labeled hairpin 2 (H2-Fc). Eventually, a large number of H2-Fc were captured to the electrode to produce amperometric signal for achieving signal amplification. This method showed a linear detection range from 5 fM to 50 nM with a detection limit of 0.78 fM. The proposed assay was successfully used to detect DENV type 1 in total RNA sample extracted, indicating great potential for application in early clinical diagnostic.