High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic...

Area of Science:

• Molecular Diagnostics
• CRISPR Technology
• Microfluidics

Background:

• CRISPR/Cas-based diagnostics show promise for rapid pathogen detection, including SARS-CoV-2.
• Current limitations include low throughput, integration challenges, and complex reagent preparation, hindering practical application.

Purpose of the Study:

• To develop an ultrahigh-throughput microfluidic system for rapid detection of SARS-CoV-2 and its variants.
• To address limitations of existing CRISPR-based diagnostic methods in terms of throughput and complexity.

Main Methods:

• A microfluidic multiplate system named mutaSCAN was developed.
• The system integrates CRISPR/Cas12a with nonextraction RT-LAMP and a deep-learning enabled prototype device.
• Self-developed reagents were utilized for enhanced performance.

Main Results:

• The mutaSCAN system detected SARS-CoV-2 in mock samples within 30 minutes at concentrations as low as 250 copies/mL.
• Achieved a throughput of up to 96 samples per round.
• Demonstrated high accuracy in clinical specimens: 98% for routine testing and 100% for mutation testing, with no false positives.

Conclusions:

• The mutaSCAN system provides a rapid, sensitive, and high-throughput solution for SARS-CoV-2 and variant detection.
• This technology overcomes key hurdles for practical, resource-limited molecular diagnostics.
• The system shows significant potential for infectious disease surveillance and mutation tracking.