A CRISPR/Cas13a-based and hybridization chain reaction coupled evanescent wave biosensor for SARS-CoV-2 gene detection†

Abstract

Timely and accurate diagnosis of RNA viruses, represented by the SARS-CoV-2, is the foundation for protecting people from health threats. Currently, direct detection of viral RNA genes remains the most accurate method. Herein, a rapid, ultrasensitive, and no heating equipment required CRISPR/Cas13a based evanescent wave fluorescence biosensing platform for quantitative detection of the SARS-CoV-2 gene is reported. The collateral effect of CRISPR/Cas13a for RNA is combined with a self-driven enzyme-free hybridization chain reaction (HCR) as a signal amplification step. When the initiator RNA strand is cleaved by Cas13a, the downstream signal amplification induced by HCR is blocked, and a multiple crRNA strategy is used to enhance the cleavage efficiency. The newly designed HCR assemblies are captured by the cDNA-modified optical fiber and generate a higher-intensity fluorescence signal induced by the evanescent field. The CRISPR/Cas13a-HCR evanescent wave fluorescence biosensing platform is capable of detection of SARS-CoV-2 with a LOD of 0.47 copies per μL and the detection time is within 35 min. The spike recovery tests in saliva and high specificity have demonstrated the potential of this method for point-of-care diagnosis. This method is also suitable for the detection of other RNA viruses, without the need to alter any design of the HCR component, and only the corresponding crRNA needs to be replaced.