Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.
Biosens Bioelectron. 2012 Mar 15;33(1):88-94. doi: 10.1016/j.bios.2011.12.024. Epub 2012 Jan 8.
An integrated microdevice of a reverse transcription-polymerase chain reaction (RT-PCR) reactor and an immunochromatographic strip was constructed for colorimetric detection of gene expression of influenza A virus subtype H1N1. An RT-PCR cocktail, which included Texas Red-labeled primers, dNTP including biotin-labeled dUTP, and RNA templates of influenza A H1N1 virus, was filled in the PCR chamber through the micropump, and the RT-PCR was performed to amplify the target H1 gene (102 bp). The resultant amplicons bearing biotin moieties and Texas Red haptens were subsequently eluted to the immunochromatographic strip, in which they were first conjugated with the gold nanoparticle labeled anti-hapten antibody in the conjugation pad, and then captured on the streptavidin coated test line through the biotin-streptavidin interaction. By observing a violet color in the test line which was derived from the gold nanoparticle, we confirmed the H1N1 target virus. The entire process on the integrated microdevice consisting of a micropump, a 2 μL PCR chamber, and an immunochromatographic strip was carried out on the portable genetic analyzer within 2.5h, enabling on-site colorimetric pathogen identification with detection sensitivity of 14.1 pg RNA templates.
一种逆转录-聚合酶链反应（RT-PCR）反应器和免疫层析条的集成微器件被构建用于甲型 H1N1 流感病毒基因表达的比色检测。RT-PCR 混合物通过微泵填充到 PCR 室中，其中包含 Texas Red 标记的引物、包含生物素标记的 dUTP 的 dNTP 和甲型 H1N1 病毒的 RNA 模板，并进行 RT-PCR 以扩增靶 H1 基因（102 bp）。带有生物素部分和 Texas Red 半抗原的所得扩增子随后被洗脱到免疫层析条上，在那里它们首先与结合垫中的金纳米颗粒标记的抗半抗原抗体结合，然后通过生物素-链霉亲和素相互作用被捕获在带有链霉亲和素的测试线上。通过观察测试线上源自金纳米颗粒的紫色，我们确认了 H1N1 靶病毒。包含微泵、2 μL PCR 室和免疫层析条的集成微器件的整个过程在便携式基因分析仪上在 2.5 小时内完成，实现了现场比色病原体识别，检测灵敏度为 14.1 pg RNA 模板。
