A highly sensitive, real-time centrifugal microfluidic chip for multiplexed detection based on isothermal...

Area of Science:

• Biotechnology
• Microfluidics
• Molecular Diagnostics

Background:

• Nucleic acid detection requires high sensitivity and multiplexing capabilities.
• Recombinase polymerase amplification (RPA) offers isothermal amplification but faces challenges with reagent viscosity and sensitivity.

Purpose of the Study:

• To develop a real-time centrifugal microfluidic chip for enhanced nucleic acid detection.
• To optimize RPA-based detection sensitivity through active mixing and two-stage amplification.

Main Methods:

• A centrifugal microfluidic chip was designed for real-time RPA.
• Optimization involved active mixing via centrifugal actuation and a two-stage amplification strategy.
• Four amplification modes were compared: with/without mixing and with/without two-stage amplification.

Main Results:

• Two-stage amplification coupled with active mixing significantly improved detection sensitivity.
• The developed microfluidic chip successfully performed multiplexed detection of African swine fever virus (ASFV) genes.
• Optimized modes demonstrated superior performance compared to standard RPA.

Conclusions:

• The integrated microfluidic system enhances RPA sensitivity and efficiency for nucleic acid detection.
• This approach provides a sensitive and parallel detection platform for pathogens like ASFV.