ABSTRACT
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
Subject(s)
COVID-19 , Nucleic Acids , Humans , RNA , CRISPR-Cas Systems , SARS-CoV-2 , RNA, Viral , Nucleic Acid Amplification TechniquesABSTRACT
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 â³sample-to-answerâ³ 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.