【作者】 丁雄；

【导师】 山崎伸二； 石磊； 马学军；

【作者基本信息】 华南理工大学 ， 制糖工程， 2014， 硕士

【摘要】 传染病是我国发展比较严重的疾病之一，如手足口病、艾滋病以及新近流行的人感染H7N9禽流感等。由于现阶段仍无有效的疫苗和特效药物，对病原的核酸检测成为预防和控制传染病的重要手段。目前，我国推荐传染病病原的核酸检测均是以PCR技术作为平台，但该法在检测上耗时耗力或依赖于昂贵笨重的仪器，不利于在基层检疫或医疗机构中的应用。相比而言，以环介导等温扩增（LAMP）为代表的新方法因检测简单、快速以及高特异性高灵敏性，有着很强的基层应用潜力。然而，LAMP法受到专利的严格保护，导致现有检测试剂盒的实际价格相对偏高，制约了真正应用。于此，本研究发明了一种新型的等温扩增技术即等温多自配引发扩增（IMSA）。本研究基于IMSA技术，分别以实时浊度法和肉眼颜色判定法两种检测方式，建立了对手足口病原EV71和CVA16、人甲型流感H7N9和艾滋病病原HIV-1的快速检测。首先，针对EV71和CVA16的VP1基因、H7N9的H7基因和N9基因以及HIV-1的gag基因的保守序列，分别设计IMSA引物；紧接着，对IMSA的反应条件进行优化；然后，验证IMSA法的检测特异性以排除交叉反应；再就，利用人工构建模板对IMSA法的灵敏度进行评估，并与现有LAMP法进行对比。最后，将所建IMSA法应用到临床样本检测中，以进一步评估检测性能。研究结果表明，优化后的IMSA法具备很强的检测特异性，无交叉反应。在检测灵敏度上IMSA法不仅可媲美现有LAMP法，甚至具备比后者更高的最低检测线。这一结论在临床样本的检测上再次得到验证。同时，本研究详细论述了IMSA法，并探索了不同引物组合的扩增效果。结果显示，IMSA只有六条引物共同使用时才能表现出快速、高特异性高灵敏性的特点。而且，本研究通过产物酶切间接证明了IMSA反应的真实存在。总之，IMSA技术作为一种新型等温扩增技术，一方面打破了LAMP专利在我国的应用限制，另一方面也是对目前核酸体外扩增技术手段的改进和补充。它具备简单、快速、高灵敏性高特异性优势，能为基层传染病防控提供新的技术思路和选择，而且具备向食品安全检测、转基因成分检测以及生态物种保护等领域扩展的潜力。更多还原

【Abstract】 Infectious disease in China develops as one of rather serious diseases, such as hand, footand mouth disease (HFMD), acquired immune deficiency syndrome (AIDS), and the newlypopular human-infected influenza A (H7N9). Since no vaccine or antiviral drugs are currentlyavailable, the rapid nucleic acid-based detection of infectious pathogens is critical for preventionand control of infectious diseases. Presently, the recommended platform for nucleic acid-baseddetection in China is polymerase chain reaction (PCR) technology. However, PCR is either time-consuming or dependents on expensive and cumbersome equipment, which is not suitable forthe application to primary level quarantine station or medical institution. In comparison withPCR, the new detection like loop-mediated isothermal amplification (LAMP) has a strongpotential in the application of primary settings, because of simple and rapid detection with highspecificity and sensitivity. Whereas, LAMP method is well protected by patents, which causesthe actual price of existing detection kits relatively high and restricted its real application. In thisregard, a novel isothermal multiple-self-matching-initiated amplification (IMSA) wasdeveloped in the study.To rapidly detect human enterovirus71(EV71) and coxsackievirus A16(CVA16) ofHMFD causative agents, human immunodeficiency virus type1(HIV-1) of AIDS, and H7N9virus, both real-time and visual IMSA assays were established. Firstly, the highly conservedregions of VP1gene for EV71and CVA16, H7and N9gene for H7N9, and gag gene for HIV-1were selected to design the corresponding IMSA primers. After optimizing the reactionconditions, the specificities and sensitivities of IMSA assays were then evaluated with theartificial templates, and the reported LAMP methods were chose as parallel tests. Finally, all theestablished IMSA approaches were applied to detect clinical specimens for further evaluation.The test results indicated that the IMSA approaches possessed high specificities without cross-reactions observed. On detection sensitivity, IMSA methods were not only comparable to theexisting LAMP methods, but also even displayed higher detection limits than the latter, whichwas further confirmed in clinical assessment. Furthermore, in the study, the details of IMSAprinciple were introduced, and the reaction effect assays with different primer combinationswere conducted, indicating that the normal six-primers set was necessary to realize simple, fast,highly specific and sensitive detection. Additionally, the IMSA reaction was indirectly provedthrough enzyme digestion experiments.In conclusion, IMSA technology as a new type of isothermal amplification technology, onthe one hand, breaks application restrictions of the LAMP patent in China; on the other hand, it makes improvement and complementarity for nucleic acid-based amplification in vitro. Due tothe advantages of simplicity, rapidness, high sensitivity and specificity, IMSA approach can beas a new technical idea and choice for the grass-roots level settings, and its application can beenlarged to other fields such as food safety inspection, detection of genetically modifiedingredients and ecological conservation.更多还原