Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge...

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• Sensitive and specific nucleic acid detection is vital for point-of-care pathogen testing, genotyping, and disease monitoring.
• Existing methods often require complex procedures and longer turnaround times, limiting their utility in resource-limited settings.

Purpose of the Study:

• To develop a novel, rapid, one-pot Cas12b-based method for multiplex nucleic acid detection.
• To integrate reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12b cleavage for enhanced efficiency.
• To validate the method's performance for point-of-care applications, including pathogen identification and mutation detection.

Main Methods:

• A one-pot assay combining RT-LAMP amplification and Cas12b collateral cleavage was designed.
• A "Green-Yellow-Red" strategy was employed for multiplex detection with visual or automated analysis.
• A portable cartridge was developed for integrated heating and fluorescence detection for point-of-care use.

Main Results:

• The method achieved complete detection within 1 hour, demonstrating high specificity and sensitivity down to single-molecule levels.
• Clinical evaluation showed a 100% positive detection rate for SARS-CoV-2 compared to RT-PCR.
• The assay successfully identified SARS-CoV-2 variants and detected low-frequency mutations (0.5%) with high accuracy.

Conclusions:

• The developed Cas12b-based one-pot assay offers a rapid, sensitive, and specific solution for multiplex molecular diagnostics.
• The "Green-Yellow-Red" strategy and portable device facilitate easy point-of-care application.
• This technology holds significant potential for diverse applications, including infectious disease surveillance and personalized medicine.