An all-in-one assay based on CRISPR/Cas13a and a DNA circuit for rapid and ultrasensitive detection of Echovirus 11

Abstract

Echovirus 11 (ECHO 11) is a positive-stranded RNA virus. The infections of ECHO 11 are associated with many clinical syndromes, thus the deployment of rapid point-of-care diagnostics is necessary. This study reported a new diagnostic assay based on All-In-One CRISPR/Cas13a and a DNA circuit (termed AIOC) for ECHO 11 detection. By combination of CRISPR/Cas13a and catalytic hairpin assembly (CHA) circuit in one-pot reaction, multiple handling steps and uncapping contamination could be effectively avoided. After recognition of target RNA, the activation of CRISPR/Cas13a induced the RNase activity of Cas13a to cleave hairpin probes, resulting in subsequent CHA amplification. This simplified assay was able to detect ECHO 11 RNA in the range of 500 × 10-18 M to 5 × 10-9 M for VP1 gene and 100 × 10-18 M to 5 × 10-9 M for RdRP gene. And the limit of detection was 152 × 10-18 M and 24 × 10-18 M, respectively. More importantly, the practicability of AIOC assay was validated by clinical ECHO samples. Taking together, the AIOC assay is rapid (∼30 min), isothermal (37 ℃), ultrasensitive, specific, user-friendly and does not need the step of RNA reverse-transcription, making it an excellent detection performance in one-pot point-of-care assay.