Nanotechnology

Chlamydia trachomatis

Loop-mediated isothermal amplification

Polymerase

Microfluidics

Biology

Gene

Molecular beacon

Oligonucleotide

Recombinase Polymerase Amplification

Molecular biology

Virology

Materials science

Nucleic acid

Computational biology

Multiplex

CRISPR

Nucleic Acid Amplification Tests

Polymerase chain reaction

Genetics

Multiplex polymerase chain reaction

Nature Communications

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 mi crofluidic device with CRISPR- C as12a a nd multiplex r ecombinase 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.

Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification