A rapid, multiplex, one-pot CRISPR/Dx system for visual detection of influenza A, influenza B, and respiratory syncytial viruses.

The detection of Influenza A Virus (IAV), Influenza B Virus (IBV), and Respiratory Syncytial Virus (RSV) presents significant diagnostic challenges due to the high similarity of clinical symptoms with other respiratory infections, leading to the need for multiplexed, rapid testing. Herein, we have established a one-pot, multiplex CRISPR/Dx detection system based on reverse transcription-recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a. It completes the detection within 30 minutes at a constant temperature of 40 °C, with the ability to detect 10 copies per µL of IAV, 10 copies per µL of IBV, and 8 copies per µL of RSV. No cross-reactivity between these respiratory viruses was observed. When equipped with our customized miniature device, it allows visual fluorescence readout without specialized equipment. Compared with conventional RT-qPCR and two-tube RT-RPA-CRISPR/Cas12a approaches, this one-pot detection system offers a simplified workflow and shorter detection time and enables visual detection, making it especially suitable for point-of-care testing and field deployment. In essence, our CRISPR/Dx system provides a novel and practical molecular diagnostic strategy for rapid and multiplex detection of respiratory pathogens to improve patient management, rational antiviral use, and epidemic control.