基于通用引物等温扩增和磁性颗粒介导聚合技术的高通量可视化个体核酸信息检测方法研究

Development of human genetics and molecular diagnostic methods have brought the advances in personalized medicine. Personalized medicine refers to the tailoring of medical treatment to the individual characteristics of each patient, implying that unique treatments can be designed for each individual. Accurate personalized nucleic acid identification is the basis of personalized medicine. Currently,clinical application eagerly need a novel individual nucleic acid detection platform with high-throughput, high-sensitivity,high-accuracy, closed detection and fast speed characteristics, and easy to operate in different level of medical and health institutions.The goal of this subject is to develop a new individual molecular diagnostic method on the basis of isothermal amplification, nanotechnology and microarray. Firstly, magnetic beads coupled with capture probes were used to enrich the effective target DNA from the sample.Then the targets were amplified by solid-phase rolling circle amplification (RCA) on the microarray printed with universal primers. Finally, magnetic beads were added on the microarray for magnetic beads mediated aggregation. Magnetic beads would physically combine with long stranded DNA and form interwined aggregates which can be detected by naked-eye.The novel method described here has great potential applications in individual SNPs genotyping and pathogen identification from various sample sources. Without the limitation of environment and infrastructure, this method can be easily used in basic medical and health epidemic prevention institutions.The implementation of this subject will provide a reliable and competitive tool for the development of personlized medicine in our country and fill the blank of domestic.

随着基因组学和分子检测技术研究不断取得进展，使得根据每个病人的个体特征提供量体裁衣式的医学诊疗服务，实现个体化医疗成为可能。准确的个体化核酸信息检测是实现个体化医疗的技术基础。临床上迫切需要一种高通量、高灵敏、准确、快速、全封闭且适用于各层级医疗卫生机构的个体化核酸检测方法。本课题中，我们将等温扩增、生物芯片和纳米技术相结合提出了新的实用性个体化核酸分子检测策略。首先利用磁性颗粒富集有效靶分子，然后利用固定有通用引物的检测芯片对靶分子进行固相多重滚环扩增，最后使用磁性颗粒介导团聚实现信号可视化检测。该方法能够同时应用于个体化SNP分型和高灵敏病原体检测。整个检测过程操作简单，且不受环境和基础设施的限制。本课题的实施将为我国个体医疗的发展提供具有国际竞争力的可靠工具，填补国内的相应空白。

随着基因组学和分子检测技术研究不断取得进展，使得根据每个病人的个体特征提供量体裁衣式的医学诊疗服务，实现个体化医疗成为可能。准确的个体化核酸信息检测是实现个体化医疗的技术基础。临床上迫切需要一种高通量、高灵敏、准确、快速、全封闭且适用于各层级医疗卫生机构的个体化核酸检测方法。本课题中，我们将等温扩增和纳米技术相结合提出了一种新型、实用性高的个体化核酸分子检测策略。首先利用磁性颗粒富集有效靶分子，然后利用通用引物对靶分子进行超支化多重滚环扩增，最后使用磁性颗粒介导团聚实现信号可视化检测。该方法能够同时检测8个分子靶标，检测灵敏度达到10nmol/uL，检测时间小于1.5小时，可以直接目视检测结果，整个检测过程操作简单，且不受环境和基础设施的限制。在此项研究的基础上，我们还构建的多重PCR结合编码磁性微粒芯片的检测方法研究。应用编码磁性微粒芯片检测5种大肠埃希氏菌，检测特异性好，检测灵敏度高，可达1.7 CFU/m。本课题的实施将为我国个体医疗的发展提供具有国际竞争力的可靠工具，填补国内的相应空白。