Thermo-Responsive Polymer Capsules in Real-Time One-Step RT-PCR for Highly Multiplex RNA Analysis

Area of Science:

• Biotechnology
• Molecular Biology
• Materials Science

Background:

• Rapid RNA detection is crucial for managing pandemic threats.
• Existing methods like RT-qPCR can produce significant by-products, affecting accuracy.
• Controlled release systems offer potential for improved RNA analysis.

Purpose of the Study:

• To develop a high-fidelity, one-step RT-qPCR system for rapid RNA detection.
• To suppress by-products in RNA amplification using thermo-responsive materials.
• To enable multiplex analysis of multiple RNA targets efficiently.

Main Methods:

• Fabrication of thermo-responsive nanocapsules with upper critical solution temperature (UCST) material and PCR primers.
• Integration of UCST nanocapsules into hydrogel microparticles containing RT primers.
• Selective RNA amplification via one-step RT-qPCR within microparticles, controlling primer release based on temperature.
• Development of facile fabrication processes using blending polymerization.

Main Results:

• Demonstrated suppression of severe side products by separating PCR primers from the RT process.
• Achieved reliable 18-plex one-step RT-qPCR within 30 minutes using single-color fluorescent optics.
• Successfully conducted extensible multiplex analysis of influenza virus, showcasing platform versatility.

Conclusions:

• The developed one-step RT-qPCR platform offers high-fidelity RNA analysis with suppressed by-products.
• The system enables rapid and simple multiplex detection of multiple RNA targets.
• The thermo-responsive nanocapsule and hydrogel microparticle system presents a versatile platform for various RNA analysis applications.