本文選題：白地霉 + 林生地霉　； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:臨床上致病性地霉有林生地霉和白地霉等,林生地霉(Geotric hum silvicola)為新近發(fā)現(xiàn)的一種罕見(jiàn)地霉,首次從巴西果蠅和印度柞蠶幼蟲(chóng)身上分離出來(lái)[1]。2002年,我科從一例膿癬患兒的皮損中分離得到一株林生地霉,首次證實(shí)該菌可以感染人類(lèi)[2]。2005年,我科又自一例中毒性表皮壞死松解癥患者的血液中分離出一株林生地霉[3],2006年黃文明[4]等從足部潰瘍中再次分離處一株林生地霉,說(shuō)明本菌既能引起皮膚地霉病,也可導(dǎo)致系統(tǒng)性感染。白地霉(Geotrichum candidum)是地霉屬的經(jīng)典代表菌種,白地霉引起的感染可侵犯呼吸系統(tǒng)、消化系統(tǒng)、循環(huán)系統(tǒng)、耳、關(guān)節(jié)及皮膚。但以支氣管感染最多見(jiàn),偶可致全身播散性感染。2007年Sfakianakis等[5]報(bào)道了1例由白地霉所致皮膚地霉病,同年我國(guó)鐘華等[6]報(bào)告了1例先天性免疫缺陷病合并播散性馬爾尼非青霉、白念珠菌及白地霉感染的病例,2008年李秀麗[7]等首次報(bào)道了白地霉引起陰道炎兩例。有關(guān)林生地霉的形態(tài)學(xué)、致病性、動(dòng)物試驗(yàn)、營(yíng)養(yǎng)生理學(xué)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體蛋白的開(kāi)發(fā)利用上,對(duì)其臨床致病性研究相對(duì)較少,鑒定仍依賴(lài)于表型特征。因此我們將從分子生物學(xué)鑒定方面對(duì)致病性地霉菌做進(jìn)一步研究。 近年來(lái),隨著科技的發(fā)展,分子生物學(xué)技術(shù)已被廣泛的運(yùn)用于真菌的實(shí)驗(yàn)室診斷,而且被證明是真菌分類(lèi)的有效方法[10]。任意引物聚合酶鏈反應(yīng)(AP-PCR)是1990年分別在不同的實(shí)驗(yàn)室同時(shí)發(fā)展起來(lái)的,已在真菌分類(lèi)鑒定和分型研究方面顯示出巨大潛力,Liu等[11]用AP-PCR法進(jìn)行真菌鑒定。本研究采用任意引物聚合酶鏈反應(yīng)(AP—PCR)對(duì)林生地霉皮損株、血液株和白地霉進(jìn)行分子生物學(xué)方面的鑒定,從而對(duì)不同菌種間的親緣關(guān)系作相似性分析,同一菌種不同部位的菌株做多態(tài)性分析,為臨床提供種間及種內(nèi)鑒定的簡(jiǎn)單、快速、可靠的方法。為探討病原微生物的傳染源、傳播途徑以及流行病學(xué)調(diào)查等提供依據(jù)。從而有利于臨床早期診斷和指導(dǎo)用藥。 方法:林生地霉皮損株與血液株為河北醫(yī)科大學(xué)第四醫(yī)院真菌室保存,均由中國(guó)科學(xué)院微生物研究所鑒定。白地霉標(biāo)準(zhǔn)菌株購(gòu)自中國(guó)科學(xué)院微生物研究所菌種保藏中心。均經(jīng)過(guò)復(fù)蘇培養(yǎng)及形態(tài)學(xué)和生化鑒定。用E.Z.N.A. Yeast DNA Kit提取地霉菌基因組DNA,采用任意引物AP3、ATG、RP2、OPA-10、S034、S040、S101、S167、S368對(duì)臨床上致病性白地霉、林生地霉皮損株和血液株的基因組DNA進(jìn)行擴(kuò)增,對(duì)各病原菌的DNA指紋的特征進(jìn)行分析。 結(jié)果:成功提取了地霉菌的基因組DNA,其純度和濃度均能滿(mǎn)足PCR反應(yīng)的要求。地霉的種間鑒定結(jié)果顯示,用九種不同引物擴(kuò)增,不同菌種間可以擴(kuò)增出大小、數(shù)目不一的DNA條帶,尤其是使用引物S040、S167、S368、AP3、OPA-10和RP2擴(kuò)增產(chǎn)生的DNA帶型種間差異較明顯。不同種真菌的DNA經(jīng)擴(kuò)增后顯示不同的DNA帶型,且同一模板在多次實(shí)驗(yàn)中均產(chǎn)生一致的DNA帶型,而且不同次提取的同種不同株真菌的DNA經(jīng)擴(kuò)增后顯示的主要DNA帶型也是相同的。分離自不同感染部位的林生地霉用隨機(jī)引物S0034、S040和S368擴(kuò)增后主要DNA帶型有明顯差異。而S167、AP3、ATG和RP2擴(kuò)增的條帶多態(tài)性均不明顯,DNA帶型基本一致。 結(jié)論:(1)采用E.Z.N.A. Yeast DNA Kit提取的地霉菌基因組DNA可以用于PCR反應(yīng)。(2)以AP3、ATG、RP2、OPA-10、S034、S040、S101、S167、S368為引物,用AP-PCR可對(duì)臨床致病性地霉從基因分子水平上進(jìn)行鑒定。AP-PCR可作為鑒定致病性地霉簡(jiǎn)單、快速、可靠的方法。(3)任意引物S040、S167、S368、AP3、OPA-10、RP2擴(kuò)增的條帶多態(tài)性都比較明顯,能把白地霉和林生地霉較好的區(qū)別開(kāi)來(lái)。(4)對(duì)于林生地霉種內(nèi)鑒定(皮損株和血液株),任意引物S034、S040和S368擴(kuò)增的條帶多態(tài)性比較明顯,DNA帶型有明顯不一致,而引物S167、AP3、ATG和RP2擴(kuò)增的DNA帶型基本一致,多態(tài)性不明顯。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R379

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