新型快速等温扩增检测miRNA方法的研究

MicroRNA(miRNA)plays important regulatory roles in many cellular processes, including differentiation, neoplastic transformation, and cell replication and regeneration. Increasing evidence indicates these biomolecules have great potential for the early diagnosis of human disease. Several unique characteristics of miRNAs, including their small size, sequence homology among family members, and low abundance in total RNA samples, make them difficult to analyze. Owing to above reasons, detecting miRNA is always low specificity，expensive, time- and labor-consuming work. Therefore, developing a high-specificity，inexpensive，simple and fast miRNA quantifiaction method becomes more urgent especially for early clinical diagnosis. Herein, we propose a reverse transcription-free approach for rapid quanti?cation of miRNAs based on the modified branch rolling circle amplification and base stacking hybridization. An exponential ampli?cation process is only carried out e?ciently in the presence of target miRNA which provides additional stability for the heterodimer of padlock probe and linking primer.This method is reverse transcription free and do not require complex apparatus in isothermal amplification which simplify the detection procedures and reduces the total analysis time and cost. In addition, the sensitivity of this method will be increased by twice using the base staking effect.For these reasons, this method is suitable for early diagnosis and clinical testing，and has broad application prospects and the significant economic and social benefits.

MicroRNA (miRNA)在细胞分化、肿瘤形成、细胞复制和再生中起着重要的调节功能。许多证据表明，这类miRNA小分子极有可能成为重大疾病早期检测的生物标记。但由于miRNA自身的特点，使其检测较难。而目前的检测方法也存在特异性不高、费用昂贵、耗时长且操作复杂等不足之处，因此开发一种准确、经济快速且操作简单的miRNA定量检测方法是非常必要的。本项目将碱基堆积原理与滚环扩增相结合，建立新型快速恒温扩增检测miRNA的方法。该方法利用miRNA的碱基堆积作用帮助挂锁引物与连接引物互补配对，使其成环，并再次利用碱基堆积力发生分支滚环扩增。由于该方法省略了传统方法中反转的过程，且整个操作过程均在恒温下进行，两次碱基堆积作用可在有效扩增的同时进一步降低非特异性扩增，提高检测的灵敏度，因此该方法适用于早期诊断和临床检测，具有广阔的应用前景和显著的经济与社会效益。

microRNA（miRNA）是一类参与了包括细胞增殖、分化、凋亡和代谢等几乎所有生命过程的小分子RNA。近些年来的研究表明 miRNA具有高度保守性，时序性和组织特异性，其异常表达往往与一些重大疾病发生和发展密切相关。因此检测miRNA表达水平有助于发现新的早期诊断和预测疗效以及患者预后的生物标志物。由于受到序列短、表达水平比较低，且有许多同源序列相似等因素的限制，miRNA的检测仍较困难。本项目以检测miRNA为目的建立了两种基于碱基堆积作用的新型检测方法。第一种方法是利用目的miRNA提供碱基堆积力使得颈环引物连接成环，进而进行滚环扩增检测，通过检测扩增产物与荧光探针杂交所获得的信号来判断miRNA的表达情况。整个过程无需反转录，且操作在恒定的温度下进行，大大的降低了检测对仪器的要求；第二种方法则是将捕获探针连接在表面被羧基化变性牛血清蛋白修饰后的上转化荧光纳米材料上，通过miRNA提供碱基堆积力使得报告探针和捕获探针能稳定的杂交获得杂交信号，并通过上转化荧光纳米在980 nm激发光的激发下所发出的荧光作为内参来达到降低系统误差准确检测的目的。这种方法能有效的区分单碱基错配的miRNA家族成员，且在较宽的浓度范围内，其检测浓度与FAM和上转化粒子的荧光比值有着较好的线性关系。综上所述，这两种方法都表现出来较好的灵敏度和特异性，由于第二种方法既无需反转录也无需扩增，且其检测浓度与FAM/UCNP的比值有着较好的线性关系，可用于定量检测，因此具有更好的实用价值，有望在疾病的早期检测和预防上发挥潜在的应用。