腺病毒分型基因芯片方法的建立与验证

发布时间：2018-05-17 11:31

本文选题：腺病毒 + 分型　；参考：《河北北方学院》2017年硕士论文

【摘要】：人腺病毒(Human adenovirus,HAdV)最早发现于人体扁桃体组织中,是一种无包膜立体对称的二十面体双链DNA病毒颗粒。腺病毒的致病谱与血清型有关,不同血清型可引起不同疾病,所以我们需要能够确定其型别的检测方法。根据文献报道,目前腺病毒有68个型别,国际病毒学分类委员会(The International Committee on Taxonomy of Viruses,ICTV)将1-52型划分为7个亚属(A-G),B亚属主要包括3、7、14、11和55型,可引起呼吸道疾病。近年来,腺病毒引起的呼吸道感染疫情时有暴发,疫情发展快、感染性强,病情严重者可危及生命,其早期检测和分型也成为医务工作者及研究者们面临的重要问题。目前的检测和分型方法中,病毒分离法耗时太长,免疫学方法并不能区分具体的血清型别。PCR扩增法通过扩增特异性基因片段,结合测序法确定其型别,但测序法灵敏度不足,主要用于实验室研究和新型腺病毒的确定。环介导等温扩增虽灵敏度高,特异性好,但其引物设计要求较高且易污染,不能实现高通量的要求。因此,迫切需要建立一种快速、简便的腺病毒分型检测方法。本文旨在建立一种腺病毒分型化学发光基因芯片检测方法,可对腺病毒3、7、14、11和55型准确分型,与病毒分离等方法相比,可缩短检测时间,提高检测效率,为腺病毒分型检测提供一种新的方法。本实验通过查阅文献确定腺病毒的靶基因,从GenBank中下载特异性基因序列,利用Primer5.0、Clustalx1.8.msw、Alignmen等生物学软件设计引物和探针,对引物和探针进行合成和筛选,制备寡核苷酸基因芯片。运用多重不对称PCR法扩增特异性基因片段,将扩增产物与基因芯片上的特异性探针杂交,经过洗涤、化学发光显色后,进行结果分析。在优化的多重PCR体系、杂交条件和化学发光检测条件下,对芯片的特异性、灵敏性和重复性进行评价,通过检测38例临床样本,考核基因芯片的实用性。本研究选取腺病毒的靶基因为hexon基因,共筛选出4对引物和5条探针,3型、7型和14型各一对引物、11和55型腺病毒共用1对引物,5种腺病毒各对应1条探针。完成了5种腺病毒质粒参考品的构建,用质粒参考品对芯片的特异性、灵敏度和重复性进行考核,以质粒为模板确定五重PCR体系进行PCR扩增,扩增产物与基因芯片上的探针杂交后,特异性良好,相互间无交叉信号,该基因芯片的最低检测限为3×103copies/μl,同一芯片内和不同芯片间的重复性变异系数CV值均小于15%。38例临床样本的分型结果与测序法结果基本一致(37/38)。综上,本文建立了一种腺病毒化学发光基因芯片检测方法,该方法可同时对3、7、11、14和55型腺病毒准确分型检测,该芯片在特异性、灵敏度和重复性方面均达到预期的性能要求,为腺病毒的临床分型诊断和流行病学调查提供了新的高通量检测方法。
[Abstract]:Human adenovirus (HAdV) was first found in human tonsils, which is an icosahedral double-stranded DNA virus particle with no capsule. The pathogenic spectrum of adenovirus is related to serotype. Different serotypes can cause different diseases, so we need to be able to determine the detection method of adenovirus type. According to the literature, there are 68 types of adenovirus. The International Committee of Classification of Virology (the International Committee on Taxonomy of virus ICTV) divides 1-52 into 7 subgenera A-GGUB, which mainly include 3C7A1411 and 55, which can cause respiratory diseases. In recent years, the respiratory tract infection caused by adenovirus sometimes breaks out, the epidemic develops rapidly, the infection is strong, the serious disease can endanger the life, its early detection and typing also become the important problem that the medical workers and researchers are faced with. In the current detection and typing methods, the virus separation method takes too long, the immunological method can not distinguish the specific serum type. PCR amplification method can amplify the specific gene fragment, combined with sequencing method to determine its type, but the sensitivity of sequencing method is not enough. It is mainly used for laboratory research and identification of new adenovirus. Although the sensitivity and specificity of the loop mediated isothermal amplification were high, the primer design requirements were high and easy to pollute, which could not meet the requirement of high throughput. Therefore, it is urgent to establish a rapid and simple method for the detection of adenovirus. The aim of this paper is to establish a chemiluminescence gene chip detection method for adenovirus-typing, which can be used to accurately type adenovirus type 3H7N14H11 and 55. Compared with the method of virus isolation, it can shorten the detection time and improve the detection efficiency. To provide a new method for the detection of adenovirus typing. In this experiment, the target gene of adenovirus was identified by consulting literature, and the specific gene sequence was downloaded from GenBank. Primers and probes were designed by Primer5.0G Clustalx1.8.mswswAlignmen and other biological software. The primers and probes were synthesized and screened, and oligonucleotide gene chips were prepared. The specific gene fragments were amplified by multiple asymmetric PCR. The products were hybridized with the specific probes on the gene chip. After washing and chemiluminescence, the results were analyzed. The specificity, sensitivity and repeatability of the microarray were evaluated under the optimized multiplex PCR system, hybridization conditions and chemiluminescence detection conditions. The practicability of the gene chip was evaluated by detecting 38 clinical samples. In this study, the target gene of adenovirus was selected as hexon gene. A total of 4 pairs of primers and 5 pairs of probes, one pair of primers, and five pairs of adenovirus probes, were selected in this study. Five kinds of adenovirus plasmid reference materials were constructed. The specificity, sensitivity and repeatability of the microarray were evaluated with the plasmid reference material. The plasmid template was used to determine the five PCR system for PCR amplification. The amplification products were hybridized with the probes on the gene chip, the specificity of the products was good, and there was no cross signal between them. The minimum detection limit of this gene chip is 3 脳 103copies/ 渭 l. The CV value of repeatability within the same chip and between different chips is less than 15.38 clinical samples. In summary, a chemiluminescence gene chip method for detection of adenovirus chemiluminescence gene chip was developed. The method can be used to detect adenovirus type 71114 and 55 at the same time. The chip achieves the expected performance requirements in terms of specificity, sensitivity and repeatability. It provides a new high-throughput detection method for clinical typing diagnosis and epidemiological investigation of adenovirus.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440;R511

【参考文献】