致病性地霉的分子生物学鉴定

发布时间：2018-05-26 01:15

本文选题：白地霉 + 林生地霉　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:临床上致病性地霉有林生地霉和白地霉等,林生地霉(Geotric hum silvicola)为新近发现的一种罕见地霉,首次从巴西果蝇和印度柞蚕幼虫身上分离出来[1]。2002年,我科从一例脓癣患儿的皮损中分离得到一株林生地霉,首次证实该菌可以感染人类[2]。2005年,我科又自一例中毒性表皮坏死松解症患者的血液中分离出一株林生地霉[3],2006年黄文明[4]等从足部溃疡中再次分离处一株林生地霉,说明本菌既能引起皮肤地霉病,也可导致系统性感染。白地霉(Geotrichum candidum)是地霉属的经典代表菌种,白地霉引起的感染可侵犯呼吸系统、消化系统、循环系统、耳、关节及皮肤。但以支气管感染最多见,偶可致全身播散性感染。2007年Sfakianakis等[5]报道了1例由白地霉所致皮肤地霉病,同年我国钟华等[6]报告了1例先天性免疫缺陷病合并播散性马尔尼非青霉、白念珠菌及白地霉感染的病例,2008年李秀丽[7]等首次报道了白地霉引起阴道炎两例。有关林生地霉的形态学、致病性、动物试验、营养生理学等各方面均已作了研究[8-9][2-4]。而白地霉目前研究大多集中在其菌体蛋白的开发利用上,对其临床致病性研究相对较少,鉴定仍依赖于表型特征。因此我们将从分子生物学鉴定方面对致病性地霉菌做进一步研究。近年来,随着科技的发展,分子生物学技术已被广泛的运用于真菌的实验室诊断,而且被证明是真菌分类的有效方法[10]。任意引物聚合酶链反应(AP-PCR)是1990年分别在不同的实验室同时发展起来的,已在真菌分类鉴定和分型研究方面显示出巨大潜力,Liu等[11]用AP-PCR法进行真菌鉴定。本研究采用任意引物聚合酶链反应(AP—PCR)对林生地霉皮损株、血液株和白地霉进行分子生物学方面的鉴定,从而对不同菌种间的亲缘关系作相似性分析,同一菌种不同部位的菌株做多态性分析,为临床提供种间及种内鉴定的简单、快速、可靠的方法。为探讨病原微生物的传染源、传播途径以及流行病学调查等提供依据。从而有利于临床早期诊断和指导用药。方法:林生地霉皮损株与血液株为河北医科大学第四医院真菌室保存,均由中国科学院微生物研究所鉴定。白地霉标准菌株购自中国科学院微生物研究所菌种保藏中心。均经过复苏培养及形态学和生化鉴定。用E.Z.N.A. Yeast DNA Kit提取地霉菌基因组DNA,采用任意引物AP3、ATG、RP2、OPA-10、S034、S040、S101、S167、S368对临床上致病性白地霉、林生地霉皮损株和血液株的基因组DNA进行扩增,对各病原菌的DNA指纹的特征进行分析。结果:成功提取了地霉菌的基因组DNA,其纯度和浓度均能满足PCR反应的要求。地霉的种间鉴定结果显示,用九种不同引物扩增,不同菌种间可以扩增出大小、数目不一的DNA条带,尤其是使用引物S040、S167、S368、AP3、OPA-10和RP2扩增产生的DNA带型种间差异较明显。不同种真菌的DNA经扩增后显示不同的DNA带型,且同一模板在多次实验中均产生一致的DNA带型,而且不同次提取的同种不同株真菌的DNA经扩增后显示的主要DNA带型也是相同的。分离自不同感染部位的林生地霉用随机引物S0034、S040和S368扩增后主要DNA带型有明显差异。而S167、AP3、ATG和RP2扩增的条带多态性均不明显,DNA带型基本一致。结论:(1)采用E.Z.N.A. Yeast DNA Kit提取的地霉菌基因组DNA可以用于PCR反应。(2)以AP3、ATG、RP2、OPA-10、S034、S040、S101、S167、S368为引物,用AP-PCR可对临床致病性地霉从基因分子水平上进行鉴定。AP-PCR可作为鉴定致病性地霉简单、快速、可靠的方法。(3)任意引物S040、S167、S368、AP3、OPA-10、RP2扩增的条带多态性都比较明显,能把白地霉和林生地霉较好的区别开来。(4)对于林生地霉种内鉴定(皮损株和血液株),任意引物S034、S040和S368扩增的条带多态性比较明显,DNA带型有明显不一致,而引物S167、AP3、ATG和RP2扩增的DNA带型基本一致,多态性不明显。
[Abstract]:Objective: Geotric hum silvicola is a rare earth mold found recently, which is a rare earth mold found in the clinical pathogenic mildew. It is first isolated from the larvae of Brazil fruit fly and India tussah larvae for the first time. A strain of the fungus is isolated from the skin lesions of a case of children with tinea purulent. In order to infect human [2].2005, our family separated a strain of [3] from the blood of a patient with toxic epidermal necrosis. In 2006, the Yellow civilization [4] was separated from the foot ulcers and another strain of the fungus, which showed that the bacteria could cause both skin mildew and systemic infection. The canon of the genus mildew is a typical fungus. The infection caused by mildew can invade the respiratory system, digestive system, circulation system, ear, joint and skin. However, the most common infection of bronchitis,.2007 Sfakianakis and other [5], which can cause systemic disseminated infection, reported 1 cases of skin mildew caused by Rhizopus alba, and 1 cases in the same year in China. In the case of disseminated immunodeficiency disease with disseminated non Penicillium, Candida albicans and Candida albicans, in 2008, two cases of vaginitis caused by mildew of Li Siu Li [7] were reported for the first time. The morphology, pathogenicity, animal test and nutrition physiology of the fungi were studied [8-9][2-4]., and the present study of M. More focus is on the development and utilization of its bacterial protein, relatively few clinical pathogenicity studies, and the identification is still dependent on phenotypic characteristics. Therefore, we will do further research on pathogenic fungi from molecular biological identification.
In recent years, with the development of science and technology, molecular biology technology has been widely used in laboratory diagnosis of fungi, and it has been proved that [10]. arbitrary primer polymerase chain reaction (AP-PCR) has been developed in different laboratories in 1990, and has been shown in the classification and classification of fungi. The great potential, Liu and other [11] were identified by AP-PCR method for identification of fungi. A random primer polymerase chain reaction (AP - PCR) was used to identify the molecular biology of the skin strain, the blood strain and the mildera mildera, so as to make the similarity analysis of the relationship among different strains, and the strains of the same strain in different parts of the strain were polymorphic. Sex analysis provides a simple, fast, reliable method for interspecific and intraspecific identification. It provides a basis for exploring the source of infection, the route of transmission and epidemiological investigation, which is beneficial to the early diagnosis and guidance of drug use.
Methods: the strain and blood strain of the fungi were preserved by the fungi room of the fourth hospital of Hebei Medical University. All of them were identified by the Institute of Microbiology of the Chinese Academy of Sciences. The standard strains of M. candidum were purchased from the center of the bacterial species preservation in the Institute of Microbiology of the Chinese Academy of Sciences. All of them were recovered, cultured, morphological and biochemical identification. E.Z.N.A. Yeast DNA Kit was used to extract the ground. The fungal genome DNA, using arbitrary primers AP3, ATG, RP2, OPA-10, S034, S040, S101, S167, S368, was used to amplify the genomic DNA of clinical pathogenic mildew, the skin strain and blood strain of the forest, and analyze the characteristics of the DNA fingerprint of the pathogenic bacteria.
Results: the genomic DNA of mycoderma was successfully extracted and its purity and concentration could meet the requirements of PCR reaction. The interspecific identification results showed that the size and number of DNA bands could be amplified with nine different primers, especially the DNA band species produced by primers S040, S167, S368, AP3, OPA-10 and RP2 amplification. The DNA of different species of fungi showed different DNA bands, and the same template produced a consistent DNA band in many experiments, and the main DNA bands of the DNA of different strains of different strains of the same species were the same. The random primer S of the isolates from different infection sites 0034, there were significant differences in the major DNA bands after S040 and S368 amplification, while the polymorphisms of S167, AP3, ATG and RP2 amplification were not obvious, and DNA banding patterns were basically the same.
Conclusions: (1) the genomic DNA of E.Z.N.A. Yeast DNA Kit can be used for PCR reaction. (2) AP3, ATG, RP2, OPA-10, OPA-10, S034, S040, etc. can be used as a primer to identify the clinical pathogenic mildew from the molecular level as a simple, rapid and reliable method for identifying pathogenic ground molds. (3) arbitrary The polymorphic bands of primers S040, S167, S368, AP3, OPA-10 and RP2 were all obvious, and could distinguish between the mildew and the fungi of the forest. (4) the polymorphisms of the bands in the species of the fungi (skin strain and blood strain) of the fungi were obvious, and the polymorphisms of the bands amplified by arbitrary primers S034, S040 and S368 were obvious, and the DNA bands were obviously different, and S167 primers, AP3, AP3 It was basically consistent with the DNA banding pattern amplified by RP2, and the polymorphism was not obvious.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379

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