Dual-engine amplification: Integrating catalytic hairpin assembly with CRISPR-Cas12a for ultrasensitive point-of-care testing of Chikungunya virus RNA.

Chikungunya virus (CHIKV) has emerged as a globally important arthropod-borne pathogen with rapid geographical expansion, urging high-performance yet simple diagnostic tools. Herein, we develop an isothermal dual-engine amplification biosensor by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a for ultrasensitive and rapid detection of CHIKV RNA. This strategy combines the enzyme-free isothermal amplification of CHA and the high-specificity trans-cleavage activity of CRISPR-Cas12a to realize cascade signal enhancement. By systematically optimizing reaction parameters including probe ratio, temperature, buffer, and incubation time, we achieve superior analytical performance. The biosensor exhibits a low limit of detection of 4 fM, a wide linear range from 10 fM to 1 μM (R2 > 0.98), and excellent specificity against dengue virus, Zika virus, and other common blood-borne viruses. It also shows strong resistance to interference from hemolysis, icterus, and lipemia. The whole assay can be finished within 60 min under isothermal conditions without sophisticated thermal cyclers. In clinical serum samples, this method achieves 97.5% concordance with RT-qPCR, with a sensitivity of 96.8% and a specificity of 100%. This CHA-CRISPR-Cas12a dual-amplification platform provides a sensitive, specific, rapid, and cost-effective approach for CHIKV detection and holds great promise for point-of-care testing in resource-limited settings.