基于集成核酸提取微流控芯片的禽流感病毒快速检测方法研究

To face the challenges of avian influenza outbreaks and other public health emergency incidents, there is an urgent need to find a highly sensitive, rapid and accurate method for identification of pathogen genes in both clinical and disease control. The traditional method is still time-consuming and labor-intensive, and it's also unable to meet the needs of rapid testing. For rapid identification of avian influenza virus subtype, our project will focus on developing a method for fast enrichment and extraction of nucleic acids based on microfluidic chip and real-time PCR. The function of sample pre-treatment, PCR amplification and real-time fluorescence detection will be integrated in a single microfluidic chip, and multicolor real-time solid-phase PCR technology will be introduced for rapid detection of avian influenza virus subtype. The proposed method will extremely shorten the detection time and overcome the existing problem of complex and multiple PCR primer design in traditional real-time PCR. It also has great potential to realize automation and fully closed of the whole process of nucleic acids extraction, amplification and real-time detection. This study will not only develop a simple, low sample consumption, rapid and high sensitive microfluidic multicolor real-time PCR technology integrated the function of nucleic acid extraction, but also will provide a new rapid detection method of pathogenic microorganisms for public health and safety, inspection and quarantine,and disease diagnosis, and it has broad application prospects.

面对禽流感病毒爆发等公共卫生事件的挑战，无论在临床医疗还是在疾病控制方面，迫切需要一种高灵敏、快速、准确鉴定病原体基因的方法。传统检测方法耗时长、操作复杂，无法满足快速检测的需求。针对禽流感病毒不同亚型快速鉴定的问题，本项目基于微流控芯片与实时荧光PCR技术，研究芯片上核酸快速富集与提取方法，将禽流感病毒检测中涉及的样品前处理、PCR扩增与实时荧光检测集成在一块微流控芯片上来实现；并且引入多重固相PCR和多色荧光探针技术，可对禽流感病毒亚型进行快速并行检测。该方法极大缩短了检测时间，可克服现有荧光定量PCR操作过程繁琐以及多重PCR引物设计复杂等难题，实现了检测过程的全自动与全封闭。本研究不但发展一种操作简单、样本使用量低、检测速度快、灵敏度高的集成核酸提取微流控多色荧光PCR新方法，而且将为我国公共卫生安全、检验检疫以及疾病诊断等领域病原微生物快速检测提供一种新途径，具有广阔的应用前景。

面对越来越多的公共卫生事件挑战，无论是疾病控制、临床诊疗，还是检验检疫部门都迫切需要一种快速鉴别病原微生物的新技术。针对多种病原微生物快速并行检测的难题，以禽流感病毒为研究对象，提出了基于微流控芯片和多色固相PCR技术的多种亚型禽流感病毒快速检测方法与技术。项目取得的重要研究结果如下：.1. 设计并研制了一种基于硅胶模吸附的集成核酸提取微流控芯片系统，该系统集成了核酸富集与纯化、核酸扩增及实时荧光检测功能，实现了核酸检测过程的全自动化操作。.2. 研制了集成高精度温度控制和多色荧光检测的实时荧光PCR装置样机，实现了变温过程的温度精确控制，温控精度达到0.1℃，升降温平均速度大于4℃/s，可同时进行五色荧光的检测。.3. 研究了多重固相PCR检测方法，解决了多重扩增不同引物间相互干扰的难题，增加了检测通量，可实现多目标基因的高通量并行检测。.4. 开展了系统验证实验，结果表明集成核酸提取微流控芯片上核酸提取的完整性、浓度和纯度达到了国际先进仪器的同等水平，成功实现了H5和H7型禽流感病毒的并行检测。.本项目的研究成果不仅可应用于各类病原微生物的快速检测，而且在分子诊断领域有着广泛的应用前景。在项目依托单位的支持下，已经在国家“双创”基地中科院合肥技术创新工程院开展转化工作，瞄准流感病毒快速检测的需求，研发系统的解决方案。