人乳头瘤病毒快速分型的目视化基因芯片技术研究及应用

发布时间：2018-03-03 16:07

本文选题：人乳头瘤病毒　切入点：胶体金-探针　出处：《西北大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究背景：目前已知宫颈癌的发病主要与人乳头瘤病毒(HPV)的高癌型感染有关。据统计全球每年有25万以上的妇女死于宫颈癌,宫颈癌是女性的第二位高发癌。随着每年宫颈癌和生殖器疣的患病率增加,对HPV感染的筛查有重要的意义。为了满足我国现阶段宫颈癌的防治需要,对妇女高危人群中宫颈糜烂患者和外生殖器部位尖锐湿疣的HPV感染分型和定量的流行病学调查与研究,对HPV的传播、宫颈癌的癌前诊断及预防提供重要依据,本项目的目的在于构建高通量、快速、灵敏且特异的HPV基因分型检测芯片系统。方法：本研究以目视化基因芯片为平台,引入多重不对称PCR及胶体金银染着色技术,以13种高、低危人乳头瘤病毒为模板,建立了可目视化基因分型的新方法。我们将样本病毒基因提取出来之后,利用通用引物GP5/GP6对基因组进行不对称扩增；设计两种探针,一种称之为捕获探针,在探针的5’末端进行氨基修饰,使其能和玻片连接,捕获探针用于和目的片段结合；另外一种探针称之为检测探针,在探针的3’端进行巯基修饰,使其能和胶体金形成共价连接,检测探针为非限制性引物的反义链可以和目的片段互补结合；在杂交过程中捕获探针和检测探针同时与目的片段相结合,胶体金就间接的标记到目的片段上,通过显色液银染显色,就会在芯片上出现一个可以肉眼观测的黑色或灰色斑点,通过平板扫描仪就可以对结果进行记录和分析。结果： 1.在检测中我们引入了β球蛋白基因作为系统的阳性指控。在对100例临床样本的检测种目视化芯片检测法与细胞学检测法的阳性率间无显著性差异(P0.05)。 2.检测探针浓度优化为1μM-5μM之间做为后期实验的探针最佳点样浓度。 3.在不对称引物比例上经过试验验证限制性引物和非限制性引物比例为1：20时效果最好,单链产物最多。 4.通过软件计算的数据可以得出在病毒模板稀释10-9时还有信号产生,银染时间为10min 5.稳定1 mL lOnm纳米金(A520=0.842)的巯基修饰探针的用量为0.4μg。
[Abstract]:Background: it is known that the incidence of cervical cancer is mainly related to the human papillomavirus (HPV) high carcinomatous infection. According to statistics, more than 250,000 women die of cervical cancer every year in the world. Cervical cancer is the second most common cancer in women. With the increasing incidence of cervical cancer and genital warts every year, screening for HPV infection is of great significance. In order to provide important basis for the transmission of HPV and the precancerous diagnosis and prevention of cervical cancer, the types and quantitative epidemiological investigation of HPV infection in cervical erosion patients and condyloma acuminatum of external genitalia in high-risk women were investigated. The aim of this project is to construct a high throughput, fast, sensitive and specific HPV genotyping detection chip system. Methods: using visual microarray as a platform, multiple asymmetric PCR and colloidal gold and silver staining techniques were introduced. Thirteen high and low risk human papillomavirus (HPVs) were used as templates. A new method of visual genotyping was established. After we extracted the virus gene from the sample, we used universal primer GP5/GP6 to amplify the genome asymmetrically, and designed two kinds of probes, one of which is called capture probe. The probe is modified with amino groups at the 5'end of the probe to enable it to connect to the glass slide, and the probe is captured for binding to the target fragment. The other probe is called the detection probe, which is modified with sulfhydryl groups at the 3 'end of the probe. It can be covalently connected with colloidal gold, and the antisense strand of the detection probe, which is a non-restrictive primer, can be complementary to the target fragment, and the capture probe and the detection probe can be combined with the target fragment simultaneously during hybridization. Colloidal gold is indirectly labeled to the target fragment, and a black or gray spot can be observed on the chip by silver staining, and the results can be recorded and analyzed by a flat plate scanner. Results:. 1. We introduced the 尾 globulin gene as a systematic positive charge. There was no significant difference in the positive rate between visual microarray and cytology in 100 clinical samples. 2. The detection probe concentration was optimized between 1 渭 M-5 渭 M as the best sample concentration in the later experiment. 3. When the proportion of restriction primer and unrestricted primer was 1:20, the best effect was obtained and the single strand product was the most. 4. The data calculated by the software can be obtained when the virus template is diluted 10-9, and the silver staining time is 10min. 5. The amount of thiol modified probe was 0.4 渭 g.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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