MicroRNA等温扩增检测新技术研究

MicroRNAs (miRNAs) play important regulation roles in organism and recently have been a hot area of research in analytical chemistry. Therefore, specific and sensitive detection of miRNA, and high-throughput detection of miRNA, are extensively focused by many analytical chemists. . According to the need of miRNA detection, we will develop two new detection methods of miRNA including specific and sensitive detection, and high-throughput detection, based on our studies on isothermal and homogeneous detections of nucleic acids. The specific and sensitive detection method of miRNA can directly give cDNA with no need of reverse transcriptase, exponentially amplify miRNA by two-step switches in isothermal and homogeneous experiment conditions, and finally the molecular beacon primers specifically generate fluorescence signals; High-throughput detection method of miRNA uses a stem-loop primer as molecular switch to reduce non-specific amplification. The cascade amplification reaction is triggered in the presence of miRNAs to give multiple products consisting of universal sequences and specific sequences that are coupled with the microarry to realize high-throughput detection of miRNAs. The success of this scheme will provide important reference for the sensitive and specific detection of miRNA, and the establishment of its expression profile.

在生物功能中具有重要调控功能的microRNAs（miRNAs）成为目前分析化学的热点研究领域之一。如何建立特异性识别的高灵敏miRNA检测方法和适宜于高通量的miRNA检测方法引起了分析化学家的广泛关注。.本课题针对于上述miRNA的检测需求，基于本研究小组近年来在核酸等温均相检测领域的研究积累，拟发展高灵敏特异性miRNA检测技术和高通量miRNA检测技术。前者可以不依赖于反转录酶进行cDNA合成，应用连续两步“开关”式反应，在等温和均相条件下以幂指数方式扩增miRNA，并通过茎环引物特异地产生荧光信号；后者依靠茎环状引物作为分子开关，减小多对引物之间的非特异扩增。在多个miRNA的存在下，可分别引发级联扩增反应，产生含有通用序列和特异序列的多个产物，该产物群与微列阵联用可实现多个miRNA同时检测。该课题的研究将为miRNA的灵敏特异检测和miRNA表达谱的研究提供重要的方法学参考。

miRNA在生长发育和疾病发生过程中具有重要的作用，建立miRNA高效灵敏快速的检测方法以及适宜于高通量的miRNA检测方法成为目前研究的趋势。为了进一步提高miRNA检测的速度及灵敏度，并使miRNA检测更具有实际应用性，本项目依据本课题组新发现的几种常见DNA聚合酶既具有DNA聚合酶的活性，同时又具有反转录酶活性的特性，构建了等温幂指数核酸扩增法实现对miRNA的高灵敏特异性检测，以及基于扩增和信号放大的等温高通量法实现对miRNA的高通量检测。反应简化了操作步骤，降低了实验成本。 . 通过本项目的研究，可以有助于解决现有RNA检测技术中存在的高通量、灵敏度、特异性不足及时间长以及成本高等问题，也将对RNA在实际样品中的检测具有重要的现实意义。