基于集成化纸基芯片的多重核酸即时检测技术研究

The point-of-care testing (POCT) of blood-transmitted infections related pathogens is of significant importance and is in great demand for the early diagnosis and treatment of patients, as well as control of the epidemic. The nucleic acid amplification technologies can realize highly sensitive detection of the pathogens, but rely on laborious operations, time-consuming steps and bulky instruments. Paper-based analytical devices (PADs) simplify the processes and operations, but yield to false positive results, due to incomplete extraction and purification of nucleic acids. This project will construct a detection platform based on integrated 3D-PADs and specific capture of nucleic acids for the genus- and species-specific diagnostic assay of malaria. The 3D-PADs integrate the capture, ligation and amplification reaction of nucleic acid detection, which simplify the steps and reduce the time. The detection method based on the specific capture of target biomarkers allow simple and selective assay, which address the problem of false positive and is free of extraction and reverse transcription of RNA. The development of the highly sensitive, selective and rapid POCT for multiple nucleic acids will provide medication guidance and treatment monitoring, as well as novel technologies to the prevention of blood-transmitted infections.

血液传播病原微生物的现场快速检测，对疾病感染者的早期诊断和治疗，以及疫情的控制至关重要，现有的核酸扩增技术虽然可以实现高灵敏度的检测，但大多操作复杂、耗时，且需要大型的仪器或设备，以纸基芯片为载体的检测平台简化了核酸检测的操作和流程，但容易因为核酸提取和纯化不彻底等原因出现假阳性，造成误诊。本项目将构建基于集成化三维纸基芯片和核酸特异性捕获技术的多重核酸检测平台，并将其应用于疟疾的现场快速检测与分型。三维纸基芯片集成了核酸检测的捕获反应、连接反应和扩增反应，简化了检测流程，提高了检测速度；基于靶标分子特异性捕获的检测方法，无需核酸提取和逆转录，操作简便，且具有更高的特异性，解决了假阳性等问题。项目的开展将开拓一种高灵敏度、高特异性和快速的多重核酸即时检测技术，不仅可以为疾病感染者提供用药指导和疗效评估，还为血液传播疾病的防治提供了新的技术手段。

传播性疾病相关病原微生物的现场快速检测，对疾病患者的早期诊断和治疗，以及疫情的防控至关重要，现有的核酸检测方法大多操作复杂，耗时，且依赖于专业的实验条件。本项目构建了用于核酸即时检测的纸基芯片平台，该检测体系利用层析试纸条对靶标进行高效而特异的捕获和富集，通过LAMP扩增对靶标进行检测，检测结果以可视化的方式进行读出，检测灵敏度高，特异性强，操作便捷，不依赖于任何高精尖的设备和仪器，检测速度快。该方法对疟原虫RNA的检测灵敏度达到了20拷贝，对新冠病毒N基因的检测灵敏度达到100拷贝每毫升，整个检测过程可在1小时内完成，其检测性能优于传统的RT-PCR和RT-LAMP等方法。该检测平台为传播性疾病的快速诊断与大规模筛查提供了新的技术手段，对疫情的防控和诊治具有重要的意义。