A one-tube RT-RPA–CRISPR/Cas12a system for zoonotic virus detection

Abstract Zoonotic viruses, such as the Nipah virus (NiV), Hendra virus (HeV), Rift Valley fever virus (RVFV), and Crimean–Congo hemorrhagic fever virus (CCHFV), pose substantial threats to global public health. Early and precise detection of these pathogens is crucial for their effective prevention and control; however, there are limitations to field applications of conventional diagnostic approaches which rely on large equipment or complex operations. In this study, we developed a rapid one-tube visual detection assay for four viruses, respectively, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology. The system incorporates highly specific CRISPR RNAs that target conserved regions of the nucleocapsid (N) genes from the 4 viruses under isothermal conditions at 39 °C. To simulate clinical conditions, we constructed recombinant vesicular stomatitis viruses expressing the N gene (rVSV-N) of NiV, HeV, RVFV, and CCHFV, respectively. Results indicated that the RT-RPA–CRISPR/Cas12a system could visually detect rVSV-N in lung samples from infected mice, with a detection limit of 1 copy/μL of viral RNA, within 40 min in a one-tube system at 39 °C isothermally. Our highly sensitive, specific, and visual one-tube detection system for NiV, HeV, RVFV, and CCHFV offers significant potential for early and accurate on-site detection of these viruses.