Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids
Yan Zhang, Yanan Lin, Ruoxuan Li, Dawei Jiang, Ruiyang Cai, Gaoxing Su, Yanyan Yu, Guangbo Qu
Abstract
Pathogen nucleic acid analysis has emerged as an indispensable component of contemporary healthcare systems, serving dual roles in personalized clinical management and population-level disease surveillance. Herein, we present a novel G-quadruplex-integrated CRISPR-Cas biosensing platform that performs in a signal-on mechanism for colorimetric detection of pathogen nucleic acids in one-pot. By harnessing the unique properties of split G4 structures, we develop a universal visual probe that generates a distinct green colorimetric signal upon target recognition, which effectively couples with both Cas12 and Cas13 systems. Using the monkeypox virus (MPXV) B7R gene and a conserved respiratory syncytial virus (RSV) sequence as model targets for Cas12a and Cas13, respectively, we establish a detection workflow combining recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage, visualized through enzymatic mediated color conversion. The tube-in-tube cartridge architecture adopted in this work enables seamless integration of RPA and CRISPR-based detection within a single closed-tube system, effectively eliminating cross-contamination risks. We successfully validate the platform for detection of MPXV in environmental samples and RSV in clinical specimens, achieving a detection limit of 1 copy per test and perfect concordance with PCR methods (40/40 agreement). The colorimetric biosensing platform developed herein demonstrates rapid (<60 min) and facial performance, establishing a novel molecular diagnostic paradigm that achieves laboratory-comparable accuracy for real-time surveillance and point-of-care applications.