Rapid and efficient CRISPR-based detection of dengue virus in a single-tube.

Background: Dengue Virus (DENV) is prevalent in tropical and subtropical regions. With the projected climate change, traditional detection methods face limitations, and there is an urgent need for more accurate and efficient diagnostic techniques.

Objective: The aim is to integrate Recombinase-aided Amplification (recombinase-aided amplification, RAA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to establish a special CRISPR-Cas system for rapid, convenient, high sensitivity, and high specificity typing detection of DENV.

Methods: CRISPR RNA (crRNA) and RAA primers were designed based on the whole genome of four DENV serotypes. A single-tube assay combining RAA with CRISPR-Cas13a technology was developed after optimizing reagent concentrations.

Results: The limit of detection (LoD) of DENV types 1-4 was 10³ copies·mL -1. No cross-reaction was observed between any of the DENV serotypes and the other three flaviviruses (Zika, West Nile, and Murray Valley encephalitis). The average sensitivity of one-step method was 95.8%, and the average specificity was 96.6%. Fluorescent signal intensities demonstrated a clear dose-dependent response, with the signal increasing as the sample concentration rose. This system can effectively distinguish non-target substances. Among them, one-step method has advantages in timeliness, ease of operation, and contamination control because it runs efficiently inside a tube and does not require the lid to be removed, but its sensitivity is relatively low. The two-steps method performs well in sensitivity.

Conclusion: In this study, we developed a novel method for rapid typing and detection of DENV using RAA and CRISPR-Cas13a in a single-tube homogeneous system.