RT-RPA-assisted CRISPR/Cas12a for rapid and multiplex detection of respiratory infectious viruses based on centrifugal microfluidics

Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA) allows for convenient, rapid, and highly sensitive nucleic acid detection. However, the non-specific cleavage characteristics of Cas12a after activation during the CRISPR nucleic acid detection process limit the detection of multiple targets. In this study, we designed a pressure-assisted and centrifugal-driven microfluidic chip for detecting respiratory pathogens. This chip achieved a high-sensitivity detection of 14 targets within 30 min. By freeze-drying, the detection reagents can be stored in the natural environment for one week and then stored at low temperature with little influence on biological activity, which reduces the difficulty of storage and transportation. The chip had independent amplification chambers to avoid competitive reactions, separating the amplification and detection chambers prevented mutual interference between the RPA and the CRISPR detection processes. Thus, this design exhibited a sensitivity of 2 copies/mu L and 14 targets nucleic acids can be detected simultaneously. Compared with real-time fluorescence quantitative polymerase chain reaction (PCR) using real clinical samples of SARS-CoV-2, the new method could accurately detect the SARS-CoV-2 virus and identify the virus subtypes, which is important for infectious disease prevention and control.