集核酸纯化、多病原体LAMP反应及在线检测为一体的床旁检测芯片研制

Microfluidic-based nucleic acids(NAs) analysis is an important research direction of Miniaturized Total Analysis Systems (μTAS). Although the development of integrated microdevices for NAs purification, amplification and online detection could lead to significant advancements in point-of-care pathogen detection, the progess is slow. We developed a glass microdevice for genetic analysis system, which integrated DNA extraction with the loop-mediated isothermal amplification (LAMP) and online detection. The DNA adsorbed to glass SPE bed in the presence of high concentration of the chaotropic agent were eluted to microchamber for LAMP reaction, and the amplification results were derived from the change of the fluorescent intensity. There is always a need for multiple pathogens detection with a single sample. In this project, we therefore aimed to develop a integrated microdevice with the capability of multiple pathogens detection, which integrates the NAs purification, loop-mediated isothermal amplification of multiple pathogens and online detection, and improves the point-of-care testing for multi-pathogen screening. Meanwhile, the high sensitivity of genetic analysis on microchip could contribute to early diagnosis for infectious diseases.

基于微流控技术的核酸分析是目前微全分析系统的重要研究方向之一，尽管集核酸纯化、扩增及在线检测为一体的整合芯片可以大大促进病原体床旁/现场检测的发展，但目前这种"sample-to-answer"的整合基因分析系统仍发展缓慢。申请者研制了一种集核酸提取、环介导等温扩增及在线检测为一体的玻璃芯片系统，芯片上提取的DNA直接被洗脱至下游的核酸扩增区，反应结束后通反应室内荧光强度的改变判断扩增与否，能快速高效地完成对单一核酸序列的检测。但临床上常常需要对一个标本同时进行多病原体筛查，而目前能够实现多病原体核酸纯化及扩增、检测的整合芯片未见报道。本项目拟在已有研究的基础上进一步改进、优化芯片结构及整合方法，发展一种能够整合核酸纯化、多病原体环介导等温扩增及在线检测为一体的微流控芯片系统，为多病原体筛查的床旁/现场检测研究提供新的方向；同时，芯片上核酸分析的高灵敏度也为感染性疾病的早期诊断提供帮助。

本课题整合等温DNA扩增技术和微流控芯片技术的特色与优势，建立一种适合于基层及欠发达地区医疗卫生机构的多病原体核酸检测方法。我们选取HPV为研究对象，建立了一种可用于HPV分型快速筛查的等温多自配引发扩增技术，实现更简易明确的结果判读，适用于基层医疗机构开展HPV基因筛查。此外，我们成功构建了数字LAMP微流控芯片，能够实现HPV亚型快速、准确、特异的定量检测。本研究中建立的定性及定量检测方法，可拓展应用于其它病原体的核酸检测，具有较高的应用前景。