Microfluidics for CRISPR-based multiplexed detection of nucleic acids

Abstract

“Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a microfluidic device with CRISPR-Cas12a and multiplex recombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.”

“a Brief overview of the steps involved in the subtyping process. The collected cervical cell specimen is first thermolyzed. Then, RPA is performed to amplify the nine HPV subtypes. The amplicons are subsequently tested via the CRISPR-Cas12a system on the microfluidic device, followed by fluorescence imaging to obtain the readout. b On-chip testing principles. A 30-plexed starburst-shaped chip (SS-Chip) with one central inlet connected to 30 outlets is used. The outlets are preloaded with various Cas12a/crRNAs that recognize the relevant target HPV subtype. After the on-chip assay, the fluorescent readout at specific outlets (i.e., space coding) indicates the presence of relevant HPV subtypes in the sample.” Reproduced under a Creative Commons Attribution 4.0 International License from Xu, Z., Chen, D., Li, T. et al. Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification. Nat Commun 13, 6480.

Figures and the abstract are reproduced from Xu, Z., Chen, D., Li, T. et al. Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification. Nat Commun 13, 6480 (2022). https://doi.org/10.1038/s41467-022-34086-y under a Creative Commons Attribution 4.0 International License.

Read the original article: Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification

Pouriya Bayat