Dual-CRISPR/Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection on Automated Centrifugal Microfluidics.

ABSTRACT

The clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection can be combined with recombinase-aided amplification (RAA) to enable rapid, accurate, and early detection of SARS-CoV-2. Current CRISPR-based approaches to detecting viral nucleic acid typically require immense manual operations to transfer RPA amplicons for CRISPR detection or suffer from compromised sensitivity by mixing the competing RPA amplification and CRISPR detection. Here, we develop dual-CRISPR/Cas12a-assisted RT-RAA assay and a ″sample-to-answer″ centrifugal microfluidic platform that can automatically detect 1 copy/µL of the SARS-CoV-2 within 30 min. This chip separates the amplification (RAA) from detection (CRISPR), such that sensitivity is maximized and the time consumption is decreased by a factor of 3. For the 26 positive and 8 negative clinical SARS-CoV-2 samples, this automated centrifugal microfluidics achieved 100% accuracy compared to the gold-standard RT-PCR technique. This point-of-care test, with the advantages of being one-step, automated, rapid, and sensitive, will have a significant potential for clinical diagnosis and disease prevention.