本文選題：艾滋病 + 真菌感染��； 參考：《河南中醫(yī)學院》2014年碩士論文

【摘要】：艾滋病病毒（HIV病毒）是一種能攻擊人體免疫系統(tǒng)的病毒，能引起人體免疫機能降低，從而導致人體對一些致病性真菌易感。有統(tǒng)計表明，90％的艾滋病人死于機會感染。血液真菌感染是AIDS患者最常見的機會性感染，且有較高的發(fā)病率及死亡率。艾滋病真菌性機會感染發(fā)病常見，但缺乏快速有效的檢測產(chǎn)品。分子診斷方法具有速度快、特異性強、靈敏度高的特點，并且能夠在早期感染階段對病原體進行檢測。但是傳統(tǒng)的分子生物學技術(shù)有檢測通量低的缺陷，而基因芯片技術(shù)具備高通量檢測的優(yōu)勢，從而解決了這一難題。 目前最常見的致病菌為白色念珠菌、煙曲霉菌、新生隱球菌等。罕見的馬爾尼菲青霉菌、卡氏肺孢子菌等真菌的致病率也呈增多趨勢。本研究通過對艾滋病流行現(xiàn)狀以及艾滋病常見真菌性機會感染現(xiàn)狀分析，選取了五種常見機會性感染真菌為檢測對象，分別為白色念珠菌、煙曲霉、新型隱球菌、馬爾尼菲青霉菌、卡氏肺孢子菌。工作匯報如下。 目的：研發(fā)一種新型可視化基因芯片產(chǎn)品，可同時檢測以下幾種真菌性機會感染病原體:馬爾尼菲青霉菌、新型隱球菌、白色念珠菌、煙曲霉、卡氏肺孢子菌。為臨床提供一種快速有效的檢測方法。 方法：基于可視化生物芯片技術(shù)，分別選取白色念珠菌sap6基因、煙曲霉cyp51A基因、新型隱球菌lac1基因、馬爾尼菲青霉菌gasc基因、卡氏肺孢子菌18s基因，并設(shè)計和篩選出五種真菌的特異性引物和探針，引物進行生物素標記，將標記好的引物進行多重PCR擴增，PCR擴增產(chǎn)物與醛基玻片上預先固定好的五種真菌特異性探針進行雜交反應(yīng)。采用可視化生物芯片技術(shù)顯色，使探針呈現(xiàn)肉眼可見的信號。經(jīng)過成像儀檢測和分析，，能特異性的檢測出此五種機會性感染真菌。 結(jié)果：本實驗通過篩選最確定5對特異性引物和5條特異性探針，對多重PCR擴增體系進行優(yōu)化，并對基因芯片的檢測性能進行了考察，基因芯片檢測靈敏度可達到103copies/mL。該方法特異性好、靈敏度高并且結(jié)果肉眼可見，試驗可在5h內(nèi)完成，操作簡便。 結(jié)論：該基因芯片具有靈敏度高、通量高等特點，可以作為真菌感染臨床診斷的一種新方法，為艾滋真菌性機會感染患者的準確診斷提供一種高通量的新方法。
[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.
【學位授予單位】：河南中醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.91

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