核酸等温扩增检测技术是生物医学检测领域的重要技术，其中重组酶聚合酶扩增（RPA）和环介导等温扩增（LAMP）技术，因其反应快速、灵敏度高、操作简便的优势，得到了广泛应用。然而，目前基于RPA和LAMP技术的传统检测方法依旧存在不足。如RPA技术的核酸扩增产物往往包含引物二聚体，对后续检测特异性产生了较大影响。LAMP技术的引物设计要求高，且易出现假阳性，导致检测特异性下降，且面向真菌快速检测的应用较少。此外，基于两种技术的传统检测方法，还受到多种仪器串行联用、样品试剂消耗量大、操作人员技术水平要求高等客观因素限制。针对上述共性关键技术问题，本论文构建了分别适用于RPA和LAMP技术的核酸等温扩增多指标微流控快速检测系统，以实现核酸的快速、高灵敏度、高特异性、多指标、低成本检测。利用上述系统，本论文完成了面向细菌、寄生虫和真菌的检测应用研究，同时验证了用于病毒和癌症基因检测的可行性，主要成果与创新如下：1、开发了基于自干涉反应芯片的固相RPA反应技术。该技术消除了引物二聚体对反应结果的影响，检测灵敏度高（<1.2 copies/μL），能实现单碱基多态性分型。2、集成高光谱干涉检测技术和自干涉反应芯片，开发RPA反应高光谱干涉原位检测系统，实现了RPA反应高光谱干涉非标记检测DNA单碱基突变，光学检测灵敏度达到了1.67 nm，可以实现恶性疟原虫DNA的快速（20 min）、非标记、高灵敏度（<6 copies/reaction）、高特异性和低成本检测（约为传统方法成本的1/10）。3、集成荧光检测平台、碟式微流控芯片和LAMP技术，开发核酸等温扩增多指标微流控快速检测系统。该系统有效提高了临床检测真菌样品的效率，实现了对外阴阴道念珠菌病（VVC）相关的四种念珠菌的快速（最快可<1 h）、高灵敏度（<1 CFU/reaction）、高特异性、多指标（最高可达22个指标）和低成本检测（约为传统方法成本的1/20），在VVC临床样品的检测中表现与金标准高度一致。综上所述，本论文搭建的系统可以实现核酸的快速、高灵敏度、高特异性、多指标、低成本检测，弥补了RPA和LAMP技术的部分不足，有望用于传染病检测、癌症精准诊断等领域。
Nucleic acid isothermal amplification detection technology is important in the field of biomedical detection, among which recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) technology has been widely used, be-cause of their advantages of fast response, high sensitivity and easy operation. However, the current traditional detection methods based on RPA and LAMP still have deficiencies. For example, the nucleic acid amplification products of RPA often contain primer dimers, which have a great impact on the specificity of subsequent detection. The primer design requirements of LAMP are high, and false positives are prone to occur, resulting in a decrease in detection specificity, and there are few applications for rapid detection of fungi. In addition, the traditional detection methods based on the two technologies are also limited by objective factors such as the serial demands of multiple instruments, the large consumption of samples and reagents, and the high technical level requirements of operators. Aiming at the above-mentioned common key technical problems, this paper constructed the nucleic acid isothermal amplification multi-target microfluidic rapid detection system suitable for RPA and LAMP respectively, to achieve rapid, high sensitivity, high specificity, multi-target, and low-cost detection of nucleic acids. Using the above-mentioned system, this paper completed the application research on the detection of bacteria, parasites, and fungi, and verified the feasibility of using it for virus and cancer gene detection. The main achievements and innovations are as follows:1. Developed a solid-phase RPA reaction technology based on a self-interference reaction chip. This technology eliminated the influence of primer dimers on the reaction results, had high detection sensitivity (<1.2 copies/μL), and could realize single base polymorphism typing.2. Integrated hyperspectral interference detection technology and self-interference reaction chip to develop RPA reaction hyperspectral interference in situ detection system, realized RPA reaction hyperspectral interference label-free detection of DNA single base mutation. The optical detection sensitivity of the system reached 1.67 nm. The system realized rapid (20 min), label-free, high sensitivity (<6 copies/reaction), high specificity, and low-cost detection of Plasmodium falciparum DNA (about 1/10 of the cost of traditional methods).3. Integrated the fluorescence detection platform, disc microfluidic chip and LAMP technology to develop a multi-target microfluidic rapid detection system for nucleic acid isothermal amplification. The system had effectively improved the efficiency of clinical detection of fungal samples, and achieved rapid (fastest <1 h), high sensitivity (<1 CFU/reaction), high specificity, multiple indicators (up to 22 indicators) and low-cost detection (about 1/20 of the cost of traditional methods) of four Candida species relating to vulvovaginal candidiasis. The performance of the system of detecting clinical samples were highly consistent with the gold standard.In summary, the systems built in this paper could realize rapid, high-sensitivity, high-specificity, multi-target, and low-cost detection of nucleic acids, which made up for some of the shortcomings of RPA and LAMP technologies and were expected to be used for infectious disease detection and precise cancer diagnosis and other fields.
