Toehold驱动树形分子组装放大技术检测唾液Hp抗体的实验研究

Helicobacter pylori (Hp) is the major pathogen of chronic gastritis and peptic ulcer, and associated with the incidence of gastric cancer, while the current eradication treatment of Hp infection is inefficient and the recurrence rate is high. The rapid and accurate detection of Hp infection is the key of early diagnosis and effective prevention. However, the routing detection methods cannot fulfill the clinical needs for noninvasive diagnosis and observation of therapeutic effects. According to the theory of common mucosal immune system, the detection of specific antibodies in saliva can be applied in the diagnosis of Hp infection, while the problems of low abundance detection must be solved. Based on the previous immunoproteomics study to screen and identify the diagnostic antigen which can be used to determine the specific Hp antibodies in saliva, this project will use the specific recombinant antigen to capture antibodies, and the aptamer specific to each type of antibodies to couple with signal amplification, design single/double-stranded probes containing the specific Toehold sequence domain as the recognition element of self-assembly amplification, construct controllable, high-yielding and functional dendritic DNA self-assembly to realize the exponential amplification, develop the noninvasive visualized detection aimed to the specific Hp antibodies including IgA/M/G in saliva. This project will provide accurate and rapid method for precision diagnosis and therapeutic effect observation, but also provide theoretical guidance and technical reference for other mucosal-associated infection diseases.

幽门螺杆菌（Hp）是慢性胃炎和消化性溃疡的主要致病菌，与胃癌的发生密切相关，目前临床治疗Hp感染根除率低、易复发。快速准确检测Hp感染是及早诊断、有效防治Hp相关疾病的关键，而现有Hp检测技术尚不能满足Hp感染无创诊断与疗效监测的临床需求。根据共同粘膜免疫系统理论，唾液特异性抗体的检测可用于Hp感染的诊断，但需解决低丰度检测问题。本项目拟在前期免疫蛋白组学筛选鉴定可用于唾液Hp特异性抗体检测的诊断抗原基础上，利用特异性重组捕获抗原结合抗体分型适配体，通过设计包含特异性Toehold区的单双链探针作为自组装反应的放大识别元件，构建可控、高产率、功能化树形自组装体，以实现信号的指数放大，建立针对唾液样本Hp特异性抗体（IgA/M/G）的无创可视化检测，以期为Hp感染的精确诊断和疗效监测提供准确快速的实验室手段，亦为其它粘膜感染性疾病的无创快速诊断提供理论指导和技术借鉴。

幽门螺杆菌（Hp）是慢性胃炎和消化性溃疡的主要致病菌，与胃癌的发生密切相关，目前临床治疗Hp感染根除率低、易复发。快速准确检测Hp感染是及早诊断、有效防治相关疾病的关键，而现有Hp检测技术尚不能满足Hp感染无创诊断与疗效监测的临床需求。根据共同粘膜免疫系统理论，唾液特异性抗体的检测可用于Hp感染的诊断，但需解决低丰度检测问题。本项目拟在前期免疫蛋白组学筛选鉴定可用于唾液Hp特异性抗体检测的诊断抗原基础上，利用特异性重组抗原磁珠结合抗体分型放大检测，通过设计包含特异性Toehold区的单双链探针作为自组装反应的放大识别元件，构建可控、高产率、功能化树形自组装体，以实现信号的指数放大，建立针对唾液样本中Hp特异性抗体（IgA/M/G）的无创可视化检测方法。本课题研究内容包括核酸自组装探针与核酶功能化探针的设计筛选；构建树形自组装反应体系，通过比较两种抗体分型放大体系研究自组装信号放大规律；优化唾液标本磁珠检测的预处理流程；对新方法进行了方法学评估。本课题建立了指数放大级树形自组装抗体检测体系，及生物条形码结合发夹自组装放大抗体检测体系，实现了唾液三种Hp特异性抗体的高灵敏、特异性可视化检测。树形自组装体对唾液抗体检测的检测限分别为50fM/25fM/80fM (IgG/IgM/IgA)，不同类型抗体、以及与BSA、溶酶体等干扰物之间无交叉反应，标准回收率为96%-101.5%；生物条形码结合发夹自组装对唾液抗体检测的检测限分别为0.5pM/0.35pM/0.68pM (IgG IgG/IgM/IgA)，不同类型抗体及干扰物之间无交叉反应，标准回收率95.4%-104.6%。通过对临床唾液样本的对比检测，该方法对IgG/IgM/IgA检测的结果分别为灵敏度89.2%/70.2%/74.6%、特异性76.5%/76.2%/78.8%、阳性预测值80.1%/76.2%/70.2%、阴性预测值74.3%/72.2%/74.4%。本研究建立的基于Toehold驱动的自组装放大策略，并将其用于唾液样本三种类型Hp特异性抗体的检测，为Hp感染的精确诊断和疗效监测提供准确灵敏的实验室手段，亦为其它粘膜感染性疾病的无创快速诊断提供理论指导和技术借鉴。