Kifaro Emmanuel George, Kim Mi Jung, Jung Seungwon, Jang Yoon-Ha, Moon Sungyeon, Lee Dong-Hun, Song Chang-Seon, Misinzo Gerald, Kim Sang Kyung
Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
Department of Veterinary Microbiology, Parasitology, and Biotechnology, Sokoine University of Agriculture (SUA), Morogoro P.O. Box 3019, Tanzania.
Diagnostics (Basel). 2023 Jan 10;13(2):261. doi: 10.3390/diagnostics13020261.
Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.
自发现以来，聚合酶链反应（PCR）已成为传染病诊断和鉴定的一项重要技术。它是一种针对各种样本类型的高度灵敏且可靠的核酸（NA）检测工具。然而，粪便携带了最为丰富的微生物和生理副产物，仍然是病原体分子检测中最棘手的临床样本。在此，我们展示了水凝胶微粒作为粪便样本中病毒RNA载体的新应用，无需事先进行RNA纯化即可用于实时聚合酶链反应（qPCR）。在作为自给自足反应隔室的引物掺入网络（PIN）的每个微粒中，固定化的逆转录（RT）引物通过杂交捕获病毒RNA，并直接启动RNA的逆转录以生成互补DNA池（PIN-cDNA池）。通过使用便携式恒温装置进行简单操作，在20分钟内获得了甲型流感病毒（IAV）的PIN-cDNA池。PIN-cDNA池可在室温下储存，或直接用于为qPCR提供cDNA模板。病毒cDNA模板在随后的qPCR中可自由释放，扩增效率超过91%。该检测方法具有良好的线性、重复性，并且与商业化的病毒RNA纯化试剂盒具有相当的检测限（LoD）。作为概念验证，这项技术具有巨大的现场应用潜力，可利用粪便样本改进人类和动物传染病监测活动，而无需实验室或离心机进行样本制备。
