Thesis Advisor杜昱光
Degree Grantor中国科学院研究生院
Degree Name硕士
Degree Discipline生物工程
Keyword核酸恒温扩增, 环介导,竞争性互补配对, 微流控芯片, 现场快速检测

甲型流感病毒(A Influenza virus)属正粘病毒科,是单股分节段的负链RNA病毒。病毒基因组共有8个片段,其中血凝素(hemagglutinin,HA)与神经氨酸酶(neuraminidase,NA)两种重要的表面糖蛋白,具很高的遗传多样性,目前已发现HA亚型18种以及NA亚型11种,其中一些亚型具有高致病性。其快速变异及进化的特性为流感病毒适应进而感染人类提供了基础,自1878年造成人流感的持续流行以来,至今仍是大型流感暴发的主要病原。甲型流感的暴发与流行严重影响食品卫生安全和国际贸易,造成了严重的公共卫生安全问题。对于甲型流感病毒的分型检测方法中,目前普遍应用的免疫荧光试验及聚合酶链式反应等方法由于其操作繁琐,耗时较长,无法满足甲型流感病毒现场快速分型检测及诊断的要求。环介导等温核酸扩增技术(Loop-Mediated Isothermal Amplification,LAMP)作为一种新型的核酸快速等温扩增技术近年来发展迅速,仅需在恒定温度下即可快速、高效、特异地扩大目标核酸,简化了核酸检测的复杂程度。竞争性互补介导核酸恒温扩增(Competitive annealing mediated isothermal amplification,CAMP)是本实验室发展的一种核酸恒温扩增的方法,同LAMP优点相似,且CAMP引物设计原理更简单,所需的目标核酸片段较短,作为新一代的分子生物学检测方法,极适合于甲型流感病毒病毒的基层检测、诊断及应用。本研究针对于目前已知的甲型流感病毒18种H型别及11种N型别建立了LAMP检测及CAMP检测,并结合微流控芯片检测的方法,实现了对于甲型流感病毒高通量的快速检测,所建立的检测方法不与其他型别发生非特异性扩增,且灵敏度高,具有良好的应用前景。;Influenza A virus belongs to the family Orthomyxoviridae, whose members are characterized by an eight-segmented RNA genome of negative polarity. The hemagglutinin (hemagglutinin, HA) and neuraminidase (neuraminidase, NA) are two important glycoprotein on surface of the gene segments. There are 18 subtypes of HA and 11 subtypes of NA based on antigenic differences in the surface glycoproteins. As present in 1878 of influenza outbreak, it has the highly potential to generate global pandemics. Systemic infection of animal and human beings, food health security and significant economic losses caused by the outbreak and epidemic of influenza A.Currently, the main diagnosis testing methods of A Influenza virus are immunofluorescence test and polymerase chain reaction. Due to the complex operation, they can not meet the requirements of rapid detection and on site diagnosis of influenza A virus. Loop-Mediated Isothermal Amplification (LAMP) is a rapidly developing new nucleic acid isothermal amplification technology that can amplify the target DNA or RNA rapidly, efficiently and specifically at constant temperature of 63℃,which simplified the complexity of nucleic acid detection. Competitive annealing mediated isothermal amplification (CAMP) is an improved method by our laboratory which amplifies the nucleic acid in the constant temperature. There are similar advantages compared with LAMP. On the other hand, primer design of CAMP principle is simpler and the target DNA fragments are shorter. As a new generation of molecular biology detection methods, they are very suitable for the virus detection and diagnosis.In this study, the established LAMP and CAMP method was sucssessflully applied for the detection of pathogens such as H1-H18 and N1-N11 types of influenza a virus. Combination with microfluidic chip detection method, we realized the high flux and rapid detection for influenza a virus for rapid detection. The established methods have lots of advantges such as high sensitivity and specificity without nonspecific amplification of other types, which have a good application prospect.

Document Type学位论文
Recommended Citation
GB/T 7714
齐立斐. 甲型流感全分型现场检测技术研发[D]. 中国科学院研究生院,2018.
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