SARS-CoV-2 detection with CRISPR diagnostics

Lu Guo; Xuehan Sun; Xinge Wang; Chen Liang; Haiping Jiang; Qingqin Gao; Moyu Dai; Bin Qu; Sen Fang; Yihuan Mao; Yangcan Chen; Guihai Feng; Qi Gu; Liu Wang; Ruiqi Rachel Wang; Qi Zhou; Wei Li

The experiments above were conducted by executing RT-RAA nucleic acid amplification and CDetection separately. However, it would be best to conduct both reactions within a single tube for convenience and, more importantly, to prevent aerosol contamination which happens when the reaction mixture has to be exposed to the environment midway through the protocol. Hence, we tried to execute both the RT-RAA and CDetection concurrently within a single tube. However, the combination resulted in a drastic decrease in sensitivity ( Fig. 1c and Supplementary information, Fig. S7 ). Therefore, in order to keep both the RT-RAA and CRISPR reactions within a single tube, we executed the RT-RAA reaction within the tube while keeping the CDetection reagents within the lid of the tube for 30 minutes, following which, the CDetection reagents were spun down into the tube for nucleic acid detection, and the resultant reaction mixture was imaged for fluorescence.