艾滋病常见真菌性机会感染基因芯片筛查试剂盒的研制

发布时间：2018-05-11 23:02

本文选题：艾滋病 + 真菌感染　；参考：《河南中医学院》2014年硕士论文

【摘要】：艾滋病病毒（HIV病毒）是一种能攻击人体免疫系统的病毒，能引起人体免疫机能降低，从而导致人体对一些致病性真菌易感。有统计表明，90％的艾滋病人死于机会感染。血液真菌感染是AIDS患者最常见的机会性感染，且有较高的发病率及死亡率。艾滋病真菌性机会感染发病常见，但缺乏快速有效的检测产品。分子诊断方法具有速度快、特异性强、灵敏度高的特点，并且能够在早期感染阶段对病原体进行检测。但是传统的分子生物学技术有检测通量低的缺陷，而基因芯片技术具备高通量检测的优势，从而解决了这一难题。目前最常见的致病菌为白色念珠菌、烟曲霉菌、新生隐球菌等。罕见的马尔尼菲青霉菌、卡氏肺孢子菌等真菌的致病率也呈增多趋势。本研究通过对艾滋病流行现状以及艾滋病常见真菌性机会感染现状分析，选取了五种常见机会性感染真菌为检测对象，分别为白色念珠菌、烟曲霉、新型隐球菌、马尔尼菲青霉菌、卡氏肺孢子菌。工作汇报如下。目的：研发一种新型可视化基因芯片产品，可同时检测以下几种真菌性机会感染病原体:马尔尼菲青霉菌、新型隐球菌、白色念珠菌、烟曲霉、卡氏肺孢子菌。为临床提供一种快速有效的检测方法。方法：基于可视化生物芯片技术，分别选取白色念珠菌sap6基因、烟曲霉cyp51A基因、新型隐球菌lac1基因、马尔尼菲青霉菌gasc基因、卡氏肺孢子菌18s基因，并设计和筛选出五种真菌的特异性引物和探针，引物进行生物素标记，将标记好的引物进行多重PCR扩增，PCR扩增产物与醛基玻片上预先固定好的五种真菌特异性探针进行杂交反应。采用可视化生物芯片技术显色，使探针呈现肉眼可见的信号。经过成像仪检测和分析，，能特异性的检测出此五种机会性感染真菌。结果：本实验通过筛选最确定5对特异性引物和5条特异性探针，对多重PCR扩增体系进行优化，并对基因芯片的检测性能进行了考察，基因芯片检测灵敏度可达到103copies/mL。该方法特异性好、灵敏度高并且结果肉眼可见，试验可在5h内完成，操作简便。结论：该基因芯片具有灵敏度高、通量高等特点，可以作为真菌感染临床诊断的一种新方法，为艾滋真菌性机会感染患者的准确诊断提供一种高通量的新方法。
[Abstract]:HIV is a kind of virus that can attack human immune system. It can cause the decrease of human immune function and lead to the susceptibility of human body to some pathogenic fungi. Statistics show that 90% of AIDS patients died of opportunistic infections. Blood fungal infection is the most common opportunistic infection in AIDS patients with high morbidity and mortality. AIDS fungal opportunistic infections are common, but lack of rapid and effective detection products. Molecular diagnostic methods have the advantages of high speed, specificity and sensitivity, and can detect pathogens in the early stage of infection. However, the traditional molecular biology technology has the defect of low detection flux, while the gene chip technology has the advantage of high throughput detection, thus solving this problem. The most common pathogens are Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. The pathogenicity of rare fungi such as Penicillium marneffei and Pneumocystis carinii also increased. In this study, the prevalence of AIDS and the status of common fungal opportunistic infections were analyzed. Five common opportunistic fungi were selected as detection objects, namely Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans. Penicillium marneffei, Pneumocystis carinii. The work report is as follows. Aim: to develop a new visual microarray for the detection of fungal opportunistic pathogens: Penicillium marneffei, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus and Pneumocystis carinii. To provide a rapid and effective method for clinical detection. Methods: based on the visual biochip technique, Candida albicans sap6 gene, Aspergillus fumigatus cyp51A gene, Cryptococcus neoformans lac1 gene, Penicillium marneffei gasc gene, Pneumocystis carinii 18s gene were selected respectively. The specific primers and probes of five fungi were designed and screened, and the primers were labeled with biotin. The labeled primers were hybridized with five fungal specific probes pre-immobilized on the aldehyde glass for multiplex PCR amplification. Visual biochip technology is used to make the probe visible to the naked eye. The five opportunistic fungi can be detected by imager. Results: the multiplex PCR amplification system was optimized by screening 5 pairs of specific primers and 5 specific probes, and the detection performance of the gene chip was investigated. The sensitivity of gene chip detection was up to 103 copies per mL. The method has the advantages of good specificity, high sensitivity and visible to the naked eye. The test can be completed within 5 hours and the operation is simple. Conclusion: the microarray has the characteristics of high sensitivity and high throughput. It can be used as a new method for the clinical diagnosis of fungal infection and provides a new high-throughput method for the accurate diagnosis of patients with HIV / AIDS opportunistic infection.
【学位授予单位】：河南中医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

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