Kim Junsun, Jung Seungwon, Kim Mi Yeon, Kim Bong Kyun, Kwon Soon Hwan, Kim Sang Kyung
Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea.
Chemical and Biological Engineering, Korea University, Seoul, 02841, Republic of Korea.
Adv Healthc Mater. 2020 Apr;9(7):e1900790. doi: 10.1002/adhm.201900790. Epub 2020 Mar 5.
Rapid and simple detection of RNA targets is in high demand due to the growing threat of pandemic viruses. One-step real-time, reverse transcription-polymerase chain reaction (One-step RT-qPCR) using a controlled release system of thermo-responsive materials is developed in this paper to enable high-fidelity RNA analysis as suppressing by-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-step RT-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-step assay is now implemented in a single microparticle, multiple target RNAs can be analyzed in a simple RT-qPCR of multiple particles. Reliable 18-plex one-step RT-qPCR is successfully conducted within 30 min using single-color fluorescent optics. This work also explains the facile fabrication processes used for the thermo-responsive nanocapsules and hydrogel microparticles by the blending polymerization method. Extensible multiplex analysis of influenza virus demonstrates the versatile uses of this one-step RT-qPCR platform.
由于大流行病毒带来的威胁日益增加，对RNA靶标的快速简单检测需求很高。本文开发了一种使用热响应材料控释系统的一步实时逆转录聚合酶链反应（一步RT-qPCR），以在抑制副产物的同时实现高保真RNA分析。由上临界溶液温度（UCST）材料和PCR引物组成的纳米胶囊根据温度携带或释放引物。将UCST纳米胶囊引入含有RT引物的水凝胶微粒中，然后通过一步RT-qPCR在微粒中选择性扩增靶RNA。通过带有纳米胶囊的微粒将PCR引物与RT过程分离，可大幅抑制严重的副产物。由于现在一步测定在单个微粒中进行，因此可以在多个微粒的简单RT-qPCR中分析多个靶RNA。使用单色荧光光学器件在30分钟内成功进行了可靠的18重一步RT-qPCR。这项工作还解释了通过共混聚合法制备热响应纳米胶囊和水凝胶微粒的简便过程。流感病毒的可扩展多重分析证明了这个一步RT-qPCR平台的广泛用途。
