Reverse transcription – quantitative polymerase chain reaction (RT-qPCR) is conventionally used method to analyze oncogenes, infected DNAs, and mutated tumor associated genes. However it is hard to detect rare genetic targets since the disturbance of undesired amplification often overrides the reaction of very few targets in a myriad of interfering genes. To solve the limitation, we developed primerimmobilized network (PIN) probe RT-qPCR which can detect target RNA with high selectivity and sensitivity. To conduct PIN probe RT-qPCR with high efficiency, design of probe, concentration of immobilized probe and qPCR condition were optimized. The LOD of PIN probe RT-qPCR was 40pg per particle with 89.2% efficiency. When extremely low concentration of RNA was used, result of PIN probe RTqPCR was showed “on/off” signal. Also, target was confirmed only in the “on” particle. The interference effects by non-target PCR products were minimized by target-capturing and washing process in the RT. Using PIN probe RT-qPCR, we successfully detected target RNA in a sample which has a ratio of 1:100,000 (positive RNA : negative RNA).

中文翻译：

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基于水凝胶微粒的RT-qPCR用于高级检测BCR-ABL1转录产物的开发

逆转录–定量聚合酶链反应（RT-qPCR）通常用于分析癌基因，感染的DNA和突变的肿瘤相关基因。但是，很难检测到稀有的遗传靶标，因为不想要的扩增的干扰通常会覆盖无数干扰基因中极少数靶标的反应。为了解决这一局限性，我们开发了引物固定网络（PIN）探针RT-qPCR，可以高选择性和高灵敏度地检测目标RNA。为了高效地进行PIN探针RT-qPCR，对探针的设计，固定探针的浓度和qPCR条件进行了优化。PIN探针RT-qPCR的LOD为每粒子40pg，效率为89.2％。当使用极低浓度的RNA时，PIN探针RTqPCR的结果显示为“ on / off”信号。也，仅在“上”粒子中确认了目标。通过RT中的靶标捕获和洗涤过程，将非靶标PCR产物的干扰效应降至最低。使用PIN探针RT-qPCR，我们成功地检测出比例为1：100,000（正RNA：负RNA）的目标RNA。