Nucleic Acid Amplification-Free Digital Detection Method for SARS-CoV-2 RNA Based on Droplet Microfluidics and CRISPR-Cas13a

Anal Chem. 2023 Nov 14;95(45):16489-16495. doi: 10.1021/acs.analchem.3c02007. Epub 2023 Nov 1.

Abstract

Most of the methods currently developed for RNA detection based on CRISPR were combined with nucleic acid amplification. As a result, such methods inevitably led to certain disadvantages such as multiple operations, expensive reagents, and amplification bias. To solve the above problems, we developed a highly sensitive and specific nucleic acid amplification-free digital detection method for SARS-CoV-2 RNA based on droplet microfluidics and CRISPR-Cas13a. In this assay, thousands of monodisperse droplets with a size of 30 μm were generated within 2 min by a negative pressure-driven microfluidic chip. By confining a single target RNA recognition event to an independent droplet, the collateral cleavage products of activated Cas13a could be accumulated in one droplet. By combining the droplet microfluidics and CRISPR-Cas13a, SARS-CoV-2 RNA could be easily detected within 30 min with a detection limit of 470 aM. The performance of this assay was verified by specificity experiments and spiking and recovery experiments with human saliva. Compared with many developed methods for SARS-CoV-2 RNA detection, our method is time- and reagent-saving and easy to operate. Taken together, this digital detection method based on droplet microfluidics and CRISPR-Cas13a provides a promising approach for RNA detection in clinical diagnostics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19* / diagnosis
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Humans
  • Microfluidics
  • Nucleic Acid Amplification Techniques
  • RNA, Viral / genetics
  • SARS-CoV-2 / genetics

Substances

  • RNA, Viral