Integrated slip valve-assisted fluidic chip coupling with CRISPR/Cas12a system for nucleic acid analysis

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensor Technology

Background:

• Existing on-site nucleic acid detection platforms require improvements in device integration and multiplexing capabilities.
• There is a need for advanced platforms that enable rapid, sensitive, and simultaneous detection of multiple nucleic acid targets in the field.

Purpose of the Study:

• To develop an integrated dual nucleic acid analysis platform for simultaneous detection of pathogens.
• To enhance device integration and multiplexing capabilities for on-site nucleic acid testing.

Main Methods:

• Development of a slip valve-assisted fluidic chip integrated with a CRISPR/Cas12a system.
• Preloading of all reagents (nucleic acid extraction, loop-mediated isothermal amplification (LAMP), and CRISPR/Cas12a detection) onto the fluidic chip.
• Utilizing a duplex LAMP reaction combined with two CRISPR detection units for simultaneous analysis.

Main Results:

• Successful construction of a CRISPR/Cas12a-based dual nucleic acid analysis platform.
• Simultaneous detection of as low as 30 copies/reaction of Vibrio parahaemolyticus and 20 copies/reaction of Salmonella typhimurium.
• Visual detection of results by the naked eye under a portable ultraviolet lamp within 60 minutes.

Conclusions:

• The developed platform offers integrated nucleic acid analysis and dual detection capabilities.
• This system provides a novel solution for on-site nucleic acid analysis with fluorescence visualized results.
• The platform demonstrates high sensitivity and efficiency for simultaneous pathogen detection.