Space-coded microchip for multiplexed respiratory virus detection via CRISPR-Cas12a and RPA

Area of Science:

• Molecular Biology
• Biotechnology
• Microfluidics

Background:

• Multiple respiratory virus infections are common in patients with respiratory diseases.
• Current detection methods are often complex, time-consuming, and not suitable for on-site use.
• Rapid, low-cost, and on-site detection is crucial for clinical diagnosis and population screening.

Purpose of the Study:

• To develop a space-coded microfluidic chip (SC-Chip) for the simultaneous detection of nine common respiratory viruses.
• To design optimized recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs based on comprehensive sequence comparison.
• To enable rapid, cost-effective, and on-site multiplexed detection of respiratory viral nucleic acids.

Main Methods:

• A space-coded microfluidic chip (SC-Chip) was designed and fabricated.
• Recombinase polymerase amplification (RPA) primers and CRISPR-Cas12a crRNAs were designed for nine respiratory viruses.
• The SC-Chip integrates RPA amplification with CRISPR-Cas12a detection for simultaneous viral identification.
• A single fluorescence probe was used for detecting all nine viral targets.

Main Results:

• The SC-Chip successfully detected nine respiratory viruses, including influenza A and B, SARS-CoV-2, and RSV, in under 40 minutes.
• The assay demonstrated a minimum detection limit as low as 10^-18 M (approximately 1 copy/reaction).
• Testing on 35 clinical patient samples showed 90% sensitivity and 100% specificity.

Conclusions:

• The developed space-coded microfluidic CRISPR system provides a rapid, cost-effective, and user-friendly method for multiplexed respiratory virus detection.
• This technology holds significant potential for improving clinical diagnosis and population screening for respiratory viral infections.
• The SC-Chip offers advantages in ease of operation and rapid data acquisition for nucleic acid detection in clinical settings.