A double-platform assay for rapid and on-site detection of chikungunya virus by integrating catalytic hairpin assembly with lateral flow immunochromatography assay.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen posing a growing global threat, yet existing nucleic acid detection methods remain difficult to implement in resource-limited settings. In this study, we developed complementary catalytic hairpin assembly (CHA)-based lateral flow immunochromatography assay platforms for CHIKV nucleic acid detection by coupling CHA with time-resolved fluorescence immunochromatographic assay (TRFIA) and colloidal gold immunochromatographic assay (GICA). After systematic optimization of reaction conditions to reduce background leakage, CHA-TRFIA achieved a detection limit of 100 fM with a linear range of 100 fM to 1 μM (R2 = 0.976), whereas CHA-GICA showed a visual detection limit of 1 pM with a linear range of 1 pM to 1 μM (R2 = 0.947). Both platforms effectively discriminated CHIKV from mutant sequences and non-target viral sequences (dengue virus, Zika virus, Mayaro virus, Ross River virus, and Sindbis virus; P < 0.0001). In blinded trials using 100 pseudovirus-spiked human serum samples (50 positive and 50 negative), at 35 °C, CHA-TRFIA and CHA-GICA achieved sensitivities of 94% and 92%, with AUCs of 0.970 and 0.984, respectively; at room temperature, the corresponding sensitivities were 92% and 88%, with AUCs of 0.980 and 0.917, respectively. Specificity was 100% for both platforms under both conditions. Both platforms completed detection within 30 min. CHA-TRFIA is suitable for quantitative analysis, whereas CHA-GICA is more appropriate for rapid equipment-free visual screening, together providing a practical option for CHIKV testing in resource-limited settings.