Application of CRISPR-Cas12a/Cas13a dual enzyme cleavage technology in the differential diagnosis of Chikungunya virus and Dengue virus

Chikungunya virus (CHIKV) and Dengue virus (DENV) infections present with highly similar clinical symptoms but require distinct management strategies, highlighting an urgent need for rapid and accurate differential diagnostic methods. The current gold standard, reverse transcription quantitative real-time PCR (RT-qPCR), has significant limitations: it relies on expensive thermal cyclers, complex laboratory infrastructure, and specialized personnel, making it difficult to implement in resource-limited settings and for on-site screening. To address this technological bottleneck, this study developed a portable, instrument-independent rapid detection technology based on the CRISPR-Cas12a/Cas13a dual-enzyme cleavage system. By precisely designing specific crRNAs targeting the CHIKV E1 gene and the DENV 3′-UTR region, and integrating them with reverse transcription recombinase-aided amplification (RT-RAA) technology, we established an integrated reaction system that performs nucleic acid amplification and detection under isothermal conditions. The core mechanism of the technology is as follows: upon recognition of the corresponding viral targets, Cas12a (targeting CHIKV) and Cas13a (targeting DENV) become activated and cleave fluorescent reporters or lateral flow strip reporter probes, enabling visual detection. Gradient-diluted viral nucleic acid tests indicated that the lowest detectable concentration that generated a signal reached 10² copies/mL, which is comparable to qPCR. Clinical simulated sample validation showed 100% overall agreement with qPCR, and the system accurately distinguished single infections from co-infections.The CRISPR dual-enzyme cleavage technology established in this study effectively circumvents the reliance of qPCR on sophisticated equipment, providing a rapid, simple, and low-cost solution for CHIKV/DENV differential diagnosis. It holds significant practical value for the early prevention and control of arboviral diseases.