HPV分型检测寡核苷酸芯片的初步研究

发布时间：2018-03-06 14:32

本文选题：HPV　切入点：宫颈癌　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 背景人类乳头状瘤病毒(Human papillomavirus, HPV)属于Papovaviridae家族,是一种双链环状DNA病毒,大小约8000 bp,目前已发现了200多种亚型。HPV感染是宫颈癌发生的主要病因,持续高危型HPV的感染是癌前病变和浸润性宫颈癌发展的必要因素。由于不同亚型HPV的致病机制和致癌危险性各不相同,因此对HPV各亚型进行分型检测有重要的临床意义。目前,临床上对HPV进行分型检测主要依靠荧光实时PCR技术,但该方法检测通量低,一个反应管中只能检测一个亚型,难以满足临床上同时检测多个亚型的需要。本研究探讨了利用寡核苷酸芯片技术同时检测HPV多个亚型的可能。目的建立带有不同亚型HPV L1基因片段的哺乳动物细胞克隆;在此基础上初步探讨利用寡核苷酸芯片技术对HPV的分型检测的可能。方法 1、建立带有不同亚型HPV L1基因片段的哺乳动物细胞克隆用做标准阳性对照。 2、收集NCBI数据库中的HPV各亚型的序列信息,设计相应的探针。 3、利用基因芯片技术及荧光定量PCR方法对HPV进行分型检测。结果 1、成功克隆了30个宫颈癌相关亚型HPVL1基因片段,成功构建并筛选出6个携带有HPV各亚型L1基因片段的细胞株。 2、初步制备出可以分型检测HPV的寡核苷酸芯片并建立了相应的检测方法。结论应用重叠延伸PCR的方法,成功克隆了30个宫颈癌相关亚型HPVL1基因片段并构建6个携带有HPV各亚型L1基因片段的细胞株;初步初步制备出可以分型检测HPV的寡核苷酸芯片并建立了相应的检测方法。
[Abstract]:Background. Human papillomavirus (HPVs) belongs to the Papovaviridae family and is a double-stranded circular DNA virus of about 8000 BP in size. At present, more than 200 subtypes of HPV infection have been found to be the main etiology of cervical cancer. Persistent and high risk HPV infection is a necessary factor for the development of precancerous lesions and invasive cervical cancer. Because different subtypes of HPV have different pathogenetic mechanisms and carcinogenic risks, it is of great clinical significance to detect the subtypes of HPV subtypes. Clinical typing and detection of HPV mainly rely on fluorescence real-time PCR technology, but the detection flux of this method is low, only one subtype can be detected in a reaction tube. It is difficult to detect multiple subtypes simultaneously in clinic. In this study, the possibility of simultaneous detection of multiple subtypes of HPV using oligonucleotide chips was discussed. Purpose. The mammalian cell clones with different subtypes of HPV L1 gene were established and the possibility of using oligonucleotide microarray to detect HPV typing was preliminarily discussed. Method. 1. Mammalian cell clones with different subtypes of HPV L1 gene were used as standard positive control. 2. The sequence information of HPV subtypes in NCBI database was collected and the corresponding probes were designed. 3. Genotyping of HPV was detected by gene chip technique and fluorescence quantitative PCR. Results. 1. 30 HPVL1 gene fragments of cervical cancer related subtype were cloned successfully, and 6 cell lines carrying L1 gene fragments of HPV subtype were successfully constructed and screened. 2. The oligonucleotide chip which can be typed to detect HPV was prepared and the corresponding detection method was established. Conclusion. By means of overlapping extension PCR, 30 HPVL1 gene fragments of cervical cancer related subtypes were cloned and 6 cell lines carrying L1 gene fragments of HPV subtypes were constructed. The oligonucleotide chip which can be used to type and detect HPV was preliminarily prepared and the corresponding detection method was established.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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