A single-step duplex CRISPR assay that integrated parallelized Cas12a and Cas13a in one-pot, coupled with visual lateral flow assay, enabling simultaneous point-of-care diagnosis of human immunodeficiency virus and Treponema pallidum .

Screening for high-risk human papillomavirus (HR-HPV) is a pivotal strategy for the prevention of invasive cervical cancer, particularly in resource-constrained settings. In this study, we developed a CRISPR-based compact automated real-time detection system, termed Ccard, for rapid testing of HPV16, HPV18, and the internal reference gene GAPDH through a streamlined workflow. A simple room-temperature treatment efficiently releases nucleic acids directly from swab samples, bypassing the need for extraction. The mixture was driven through the channels and loaded into the reaction chambers on the microfluidic chip by a vacuum pump. The lyophilized reagents in the reaction zone of the microfluidic chip were redissolved. The system subsequently performs a single-step RPA-CRISPR/Cas13a reaction, with real-time detection enabled by an Internet of Things device. The assay achieved detection within 30 min, with a limit of detection (LOD) of 5 copies/µL for both HPV16 and HPV18. Validation with clinical samples demonstrated high sensitivity (96.8% for HPV16 and 100% for HPV18) and 100% specificity. These results highlight the reliability, precision, and strong potential of the Ccard system for rapid testing of HR-HPVs.