微小RNA实时荧光恒温指数扩增反应(ReFIRE)的关键技术研究

The detection technologies of microRNA (miRNA) provide indispensable technical supports for the basic scientific researches and clinical applications of non-conding RNA (ncRNA). However, the current available detection methodologies of miRNA could not satisfied the robust requirement of ncRNA researches because of the unique molecule characteristics of miRNA. In the current project, based on the combined advatages of nicking endonuclease, DNA polymerase with strand displacement activities, and fluorescence labled DNA/locked nucleic acid (LNA) chimera primers, a novel method, named as REal-time Fluorescent Isothermal Reaction with Exponential amplification (ReFIRE), was developed to simultaneous detection up to hundreds of miRNA in a single reaction units. Moreover, based on the basic principle of ReFIRE, three novel miRNA detection methods were plan to developed in the current project, i.e., low-throughput multiplex detection in a single tube, medium-throughput liquid-phase biochips, and high-throughput solid-phase biochips. All methods had abilities to disdinguish miRNAs having single base variations, respectively finish 5, 25, and 50 kinds of miRNA in just 20 minutes. The ReFIRE technologies developed in the current project has completely independent intellectual property rights. And a serial of novel miRNA detection methods which could satified various specific requirements of miRNA detection could be developed based on ReFIRE basic priciples, such as single tube analysis, liquid-phase biochips, solid-phase biochips, absolute digital quantitative analysis, and in situ analysis. Moreover, besides miRNA, the ReFIRE could also be further developed to detech other types of ncRNA molecules. In conclusion, the novel ReFIRE technologies could provide a serial of novel methodology supportments for the ncRNA researches. Because having high-thoughput properties, high sensitivitie and specifities, simple and fast characteristics, the development of ReFIRE could sharply decrease the experiment design problems associated with ncRNA researches, and therefore effectively increase the working efficiencies of ncRNA researches.

微小RNA(miRNA)检测技术是非编码RNA基础研究和临床应用不可或缺的技术保障，但现有检测技术尚不能满足miRNA独特分子属性近乎苛刻的需求。本项目利用缺口酶、DNA聚合酶、荧光标记DNA/LNA嵌合引物的协同作用优势，研发可同时检测多种miRNA的实时荧光恒温指数扩增反应(ReFIRE)，初步建立低通量单管多重检测、中通量液态芯片、高通量固态芯片等三种检测方法，三种方法的总RNA需求量仅pg级，灵敏度可达5-10拷贝/反应，可识别单碱基变异，可在20分钟内分别完成5、25和50种miRNA的平行检测。本项目拟研发的ReFIRE技术具有完全自主知识产权，可衍生出单管检测、液态芯片、固态芯片、数字化绝对定量、原位检测等满足不同需求的系列方法，与现有检测技术相比较，具有通用性好、灵敏度高、特异性强、简便实用等优势，还可应用于其它非编码RNA的检测，在非编码RNA研究领域具有良好的应用前景。

本研究系统地研究了实时荧光恒温指数扩增ReFIRE(Real-time Fluorescent Isothermal Reaction with Exponential amplification, ReFIRE)反应体系中引物、DNA聚合酶、切口酶、反应缓冲液、dNTPs等参数对ReFIRE检测灵敏度、特异性、重复性、线性范围的影响规律，优化了ReFIRE反应体系，获得了能够在介于55-65℃之间单一恒定温度条件下检测多种微小RNA（miRNA）的最佳性能指标。成功构建了基于ReFIRE原理的miRNA检测体系，实现了单个反应管中同时检测5种miRNA的ReFIRE检测方法，并成功对仅有一个或少数几个碱基差异的Let-7家族分子进行区分与鉴定，该体系具有单碱基变异识别能力，检测时间小于60 min，灵敏度可达60个拷贝/反应。用于鉴定呼吸系统相关疾病的中通量液相、高通量固相miRNA检测芯片，已经完成引物设计与修饰及引物性能检测，芯片正在制备加工与调试中。同时，在ReFIRE原理基础上，我们设计并研究了一种环形引物介导的恒温EXPAR(Exponential amplification reaction)扩增系统，通过对引物、缺口酶、荧光染料等性能指标的优化获得EXPAR环形引物扩增体系，该体系成功鉴定出乳腺癌细胞株MCF-7中miRNA21和miRNA10的相对表达量。本项目开发的一系列检测方法可以满足miRNA或ncRNA多样性和复杂性对检测方法的特殊需求，为ncRNA的基础研究和临床应用提供检测新技术和新方法。相关研究成果已发表国外SCI论文2篇，申请专利4项，获得专利授权1项，培养硕士研究生1名，博士后1名。