Thermo-responsive Polymer Capsules in Real-time One-step RT-PCR for Highly Multiplex RNA Analysis

Abstract

Rapid and simple detection of RNA targets is in high demand due to the growing threat of pandemic viruses. One&#8208

step real&#8208

time, reverse transcription&#8211

polymerase chain reaction (One&#8208

step RT&#8208

qPCR) using a controlled release system of thermo&#8208

responsive materials is developed in this paper to enable high&#8208

fidelity RNA analysis as suppressing by&#8208

products. The nanocapsules, consisting of upper critical solution temperature (UCST) material and PCR primers, carry or release the primers depending upon the temperature. The UCST nanocapsules are introduced into hydrogel microparticles incorporated with RT primers and then the target RNA is selectively amplified in the microparticle through one&#8208

qPCR. Severe side products are sharply subdued by separating the PCR primers from the RT process by means of the microparticles with nanocapsules. Because the one&#8208

step assay is now implemented in a single microparticle, multiple target RNAs can be analyzed in a simple RT&#8208

qPCR of multiple particles. Reliable 18&#8208

plex one&#8208

qPCR is successfully conducted within 30 min using single&#8208

color fluorescent optics. This work also explains the facile fabrication processes used for the thermo&#8208

responsive nanocapsules and hydrogel microparticles by the blending polymerization method. Extensible multiplex analysis of influenza virus demonstrates the versatile uses of this one&#8208

qPCR platform.