CAS OpenIR
核酸现场检测关键技术研究及初步应用
毛瑞
Subtype博士
Thesis Advisor杜昱光
2018-07
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
Keyword核酸恒温扩增,微流控芯片,现场快速检测,竞争性互补配对,Camp
Abstract

癌症和传染性疾病等仍为人类健康生活的严重威胁,而相关的生物标志物的快速、准确检测是有效应对的前提。蛋白和核酸标志物是当前体外诊断的主要目标分子,而针对蛋白检测的免疫技术一般敏感性较差,故核酸分子靶标为对象的检测技术成为主流。核酸检测中成熟的PCR及其衍生技术因严格的温控和精密仪器等需求而限制了应用场景。而以环介导恒温扩增(LAMP)为代表的新技术具有简便、快速、高特异性和高灵敏性的优势。然而,LAMP等恒温扩增技术引物设计复杂并受到严格专利保护,制约了真正的应用。同时,微流控芯片的流体精准操控及规模化集成优势可将核酸检测步骤高度集成,将明显降低人工操作步骤、缩短检测时间,其固有的微型化优势亦使得便携式现场多靶标高通量的核酸分析成为可能。本研究以基于微流控芯片的多重多靶标核酸检测和新型核酸恒温扩增技术的研发为核心,结合快速样品前处理技术基本完成了现场核酸检测关键技术体系的构建。主要内容及结果如下:(1)以微流控芯片为核心,构建了基于LAMP技术的多重多靶可视化及荧光核酸的现场核酸检测平台,实现了应用于疟疾疫情防控的6指标同时检测可视化芯片研发及应用于中东呼吸道综合征病毒(MERS-CoV)的荧光检测芯片研发,并探索配套常温储存试剂的冷冻干燥技术。(2)研发了基于竞争性互补配对的核酸恒温扩增技术(Competitive annealing mediated isothermal amplification of nucleic acids,CAMP),该技术利用混合引物的设计思路和DNA聚合酶的链置换活性,通过引物结合、延伸、解链、临近互补、再次延伸的循环,实现了恒温条件下核酸扩增的目的。该方法与LAMP技术相比,引物设计难度和目标核酸长度要求明显降低。此外,在CAMP中引入外引物和环引物可进一步提高核酸扩增速率。随后针对CAMP技术进行了反应体系中反应成分、引物设计、荧光试剂的优化,最终建立了CAMP这一新型恒温扩增方法。(3)将新研发的CAMP技术应用于甲型H1N1流感病毒和犬病毒等病原的的检测获得了成功。随后利用已建立的微流控检测平台并结合快速核酸提取技术实现了鲤疱疹病毒的微流控CAMP现场检测的技术体系构建。(4)提出并验证了另一种基于螺旋环结构的核酸恒温扩增新方法(Helix loop isothermal amplification of nucleic acids,HAMP),同CAMP一样,具有良好的应用前景。;Cancer and infectous disease still pose great threat to health, and the rapid and accurate detection of related biomarkers is the prerequisite for effective response. Protein and nucleic acid biomarkers are the main target molecules for in vitro diagnosis. However, the immunoassay based protein detection methods are generally not sensitive. Therefore, the detection technology of nucleic acid has become the mainstream of rapid detection.The PCR and its derivative techniques in nucleic acid detection have limited the application scene because of strict temperature control, and sophisiticated instruments. The new technology represented by loop-mediated isothermal amplification (LAMP) has the advantages of simple, rapid, high specificity and high sensitivity. However, the primer design of LAMP and other isothermal amplification methods are complex and subject to strict patent protection, which restricts the application. Microfluidic chip can integrate steps of nucleic acid detection by take the merits of precision fluidics control and large scale integration, which will greatly reduce the manual operation steps, shorten the detection time. And the inherent miniaturization of microfluidic chip will also make the field portable multi-target nucleic acid analysis possible.The work were centered on key technologies of on-site nucleic acids detection by multiplex microfluidic chip, invention of novel isothermal amplication and rapid sample pretreatment. The main contents and results were as followed:(1) Foundation of visually and fluorescent multiplex microfluidic detection chip based on LAMP method for on-site nucleic acid detetion. The microfluidic chip for 6 targets visually detection at once for malira prevention was developed. And the microfluidic chip for fluorescent detection of Middle East Respiratory Syndrome Coronavirus was developed. At last, the freeze-drying technology for room temperature storage reagents of LAMP was explored.(2) The Competitive annealing mediated isothermal amplification of nucleic acids (CAMP) was invented. CAMP takes the advantage of hybrid primers design and DNA polymerase with strand displacement activity. Through the cycle of primer annealing, extension, competitive annealing and extension, the purpose of nucleic acid amplification at isothermal conditions is achieved. In comparasion of LAMP, the principle of primer design is simpler and the required nucleic acid fragment is shorter in CAMP. Moreover, the outer primers and loop primers were introduced in order to improve the reaction rate of CAMP. At last, the reaction system of CAMP was optimized in compotents, primer design and fluorencent agents for the establishment of the novel nucleic acid isothermal amplification method.(3) The established CAMP method was sucssessflully applied for the detection of pathogens such as H1N1 Influenza A virus and dog virus. And the microfluidic based CAMP method coupled with rapid nucleic adids extraction was developed for the on-site detection of Koi herpesvirus.(4) In addition, another isothermal nucleic acids amplification method called helix loop mediated isothermal amplification of nucleic acids (HAMP) was aslo verified, would be of great promise for further application. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26882
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
毛瑞. 核酸现场检测关键技术研究及初步应用[D]. 中国科学院研究生院,2018.
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