發(fā)布時(shí)間：2018-06-05 05:19

  本文選題：近平滑念珠菌復(fù)合體 + 都柏林念珠菌��； 參考：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文

【摘要】：第一部分近平滑念珠菌復(fù)合體表型與基因型研究 第一章近平滑念珠菌復(fù)合體的篩選、測序鑒定和種系進(jìn)化分析 目的研究我國華東地區(qū)近平滑念珠菌復(fù)合體的菌種分布和種系進(jìn)化關(guān)系。方法利用科瑪嘉念珠菌顯色培養(yǎng)基和API20CAUX生化系統(tǒng)對分離白華東地區(qū)4個(gè)城市的80株臨床株進(jìn)行表型鑒定。經(jīng)生化鑒定為近平滑念珠菌、季也蒙念珠菌、無名念珠菌和葡萄牙念珠菌的菌株進(jìn)一步進(jìn)行ITS區(qū)測序分子鑒定,構(gòu)建基于ITS區(qū)序列的種系進(jìn)化樹,分析近平滑念珠菌復(fù)合體中菌株的種系親緣關(guān)系。結(jié)果80株臨床株中,60株菌株(75%)經(jīng)生化系統(tǒng)鑒定為近平滑念珠菌,11株(13.75%)為光滑念珠菌,4株(5%)為季也蒙念珠菌,葡萄牙念珠菌、乳酒念珠菌、涎沫念珠菌、郎比卡念珠菌和無名念珠菌各1株(1.25%)。60株表型鑒定為近平滑念珠菌中,3株分子鑒定為季也蒙念珠菌,57株菌為近平滑念珠菌復(fù)合體,其中Candida parapsilosis sensu stricto占71.9%(41/57),C. metapsilosis占28.1%(16/57)；未分離到Corthopsilosis。菌種分布呈現(xiàn)顯著的地域差異：來自南京、上海和濟(jì)南的37株臨床株中,C. parapsilosis sensu stricto占89.8%(33/37),C. metapsilosis僅占10.2%(4/37)；而來源于南昌的臨床株中則以C. metapsilosis為主,占60%(12/20)。基于ITS區(qū)的種系進(jìn)化樹表明：C. parapsilosis sensu stricto進(jìn)化上較為保守；C.metapsilosis和C. orthopsilosis種系親緣關(guān)系更為緊密；C. metapsilosis存在兩種分布不均的ITS基因型(Ⅰ和Ⅱ),以Ⅱ型為主。結(jié)論(1)近平滑念珠菌復(fù)合體在我國華東地區(qū)的菌種分布存在地區(qū)差異；(2) C. metapsilosis菌株中存在差異分布的ITS基因型。 第二章近平滑念珠菌復(fù)合體的PCR-RFLP和RAPD鑒定分型研究 目的構(gòu)建和評價(jià)用于近平滑念珠菌復(fù)合體分子鑒定的PCR-RFLP和RAPD-PCR方法,并進(jìn)一步對臨床株進(jìn)行RAPD指紋分型和聚類分析。方法(1)POM-StuI/StyI酶切法：設(shè)計(jì)近平滑念珠菌復(fù)合體特異性引物POMf/r擴(kuò)增ITS區(qū)的種間多態(tài)性片段POM, PCR擴(kuò)增POM片段,后分別用StuI和StyI進(jìn)行酶切反應(yīng)。(2)隨機(jī)引物RP02進(jìn)行RAPD-PCR擴(kuò)增臨床株基因組多態(tài)性片段,形成RAPD指紋,并對RAPD指紋進(jìn)行聚類分析。結(jié)果(1) POMf/r特異性擴(kuò)增C. parapsilosis sensu stricto, C. orthopsilosis和C. metapsilosis標(biāo)準(zhǔn)株和臨床株的ITS區(qū)POM片段,經(jīng)StuI和StyI酶切后形成種特異性RFLP條帶,與測序鑒定結(jié)果一致。(2) RP02-RAPD反應(yīng)產(chǎn)生C. parapsilosis sensu stricto和C. metapsilosis種特異性RAPD指紋圖譜；RAPD指紋聚類分析表明：C. metapsilosis存在2種差異分布的基因型,與ITS基因型相一致。結(jié)論(1) POM-StuI/StyI酶切法可以實(shí)現(xiàn)近平滑念珠菌復(fù)合體的快速而準(zhǔn)確的分子鑒定；(2)RPO2-RAPD也可以實(shí)現(xiàn)C. parapsilosis sensu stricto和C. metapsilosis快速分子鑒定；(3)'C. metapsilosis存在2種差異分布的RAPD基因型,與ITS基因型一致。 第三章近平滑念珠菌復(fù)合體藥物敏感性研究 目的測定近平滑念珠菌復(fù)合體對常見抗真菌藥物的敏感性。方法參照美國CLSIM27-A3指南進(jìn)行藥敏試驗(yàn),檢測近平滑念珠菌復(fù)合體臨床株對兩性霉素B、氟康唑、伊曲康唑、伏立康唑和米卡芬凈的敏感性。結(jié)果所有菌株對兩性霉素B、氟康唑、伏立康唑和米卡芬凈均敏感,6株C. parapsilosis sensu stricto和3株C.metapsilosis對伊曲康唑呈劑量依賴性敏感。未發(fā)現(xiàn)耐藥株。結(jié)論臨床上近平滑念珠菌耐藥株罕見,常見抗真菌藥物可以有效治療近平滑念珠菌感染。 第四章近平滑念珠菌復(fù)合體胞外水解酶研究 目的檢測34株近平滑念珠菌復(fù)合體菌株(21株C. parapsilosis sensu stricto,1株C. orthopsilosis和12株C. metapsilosis)產(chǎn)生胞外水解酶的能力。方法利用卵黃培養(yǎng)基、胎牛白蛋白培養(yǎng)基和吐溫-80培養(yǎng)基分別檢測近平滑念珠菌復(fù)合體菌株產(chǎn)生磷脂酶、蛋白酶和酯酶的能力。結(jié)果(1)磷脂酶活力：90.5%(19/21)C. parapsilosis sensu stricto和91.7%(11/12)C. metapsilosis菌株產(chǎn)生磷脂酶活力,兩種真菌酶活力無顯著性差異,C. orthopsilosis標(biāo)準(zhǔn)株未檢測到磷脂酶活力；(2)蛋白酶活力：81.0%(17/21) C. parapsilosis sensu stricto和83.3%(10/12)C. metapsilosis菌株表達(dá)蛋白酶活力,前者活力更強(qiáng)；C. orthopsilosis標(biāo)準(zhǔn)株表達(dá)極強(qiáng)水平的蛋白酶活力；(3)酯酶活力：僅有4.76%C. parapsilosis sensu stricto (1/21)和16.7%C.metapsilosis (2/12)表達(dá)酯酶活力,C. orthopsilosis標(biāo)準(zhǔn)株未表達(dá)酯酶活力。結(jié)論本研究中C.parapsilosis sensu stricto, C. metapsilosis與白念珠菌類似,均高表達(dá)磷脂酶和蛋白酶活力,但酯酶活力極低。 第二部分都柏林念珠菌的鑒定方法研究 第五章都柏林念珠菌的經(jīng)典表型方法與分子生物學(xué)鑒定方法研究 目的比較6種都柏林念珠菌經(jīng)典表型鑒定方法和構(gòu)建、評價(jià)一種新的菌落PCR-RFLP分子鑒定方法方法(1)利用科瑪嘉顯色培養(yǎng)基、玉米粉吐溫培養(yǎng)基厚壁孢子形成試驗(yàn)、45℃耐受試驗(yàn)、高滲培養(yǎng)基耐受試驗(yàn)、煙葉培養(yǎng)基形態(tài)學(xué)觀察和API20C AUX生化鑒定系統(tǒng)等6種經(jīng)典表型方法鑒定白念珠菌和都柏林念珠菌標(biāo)準(zhǔn)株和66株白念珠菌/都柏林念珠菌臨床分離株；(2)菌落PCR-ApaI/BsiEI酶切法：菌落PCR擴(kuò)增白念珠菌、都柏林念珠菌和其它8種常見致病酵母菌標(biāo)準(zhǔn)株的D1-D2區(qū),用ApaI和BsiEI分別進(jìn)行酶切鑒定；并對臨床分離株進(jìn)行分子鑒定。結(jié)果(1)高滲培養(yǎng)基耐受試驗(yàn)、煙葉培養(yǎng)基形態(tài)學(xué)觀察可以準(zhǔn)確鑒定白念珠菌和都柏林念珠菌標(biāo)準(zhǔn)株；66株臨床株經(jīng)表型鑒定均為白念珠菌；(2)菌落PCR-ApaI/BsiEI產(chǎn)生白念珠菌和都柏林念珠菌特異性RFLP帶型,66株臨床株經(jīng)分子鑒定均為白念珠菌。 結(jié)論(1)聯(lián)合2種或更多種表型鑒定方法可以實(shí)現(xiàn)都柏林念珠菌的篩選和初步鑒定；(2)菌落PCR-ApaI/BsiEI可以實(shí)現(xiàn)白念珠菌和都柏林念珠菌的快速準(zhǔn)確的分子鑒定。
[Abstract]:Part 1 phenotype and genotype of Candida albicans complex
Chapter 1 screening, sequencing and phylogenetic analysis of Candida albicans complex
Objective to study the species distribution and phylogenetic relationship of the near Candida albicans complex in East China. Methods the phenotypic identification of 80 clinical strains isolated from 4 cities in white Huadong area was identified by coloration and API20CAUX biochemical system of Candida albicans. The biochemical identification was characterized by Candida albicans, Candida albicans and nameless The strains of Candida albicans and Candida albicans were further identified in the ITS region sequence, and the phylogenetic tree based on the ITS region sequence was constructed, and the phylogenetic relationship of the strains in the Candida albicans complex was analyzed. Results of the 80 clinical strains, 60 strains (75%) were identified by biochemical system as Candida albicans, 11 (13.75%) of Candida albicans, 4 Strains (5%) were Candida albicans, Portuguese Candida, Candida albicans, Candida albicans, Candida albicans and Candida albicans (1.25%) of 1 (1.25%).60 strains were identified as near Candida albicans, 3 of which were Candida albicans and 57 strains of Candida albicans, and Candida parapsilosis sensu stricto was 71.9% (71.9%). 41/57), C. metapsilosis accounted for 28.1% (16/57), and the distribution of unseparated Corthopsilosis. strains showed significant regional differences: among 37 clinical strains from Nanjing, Shanghai and Ji'nan, C. parapsilosis sensu stricto accounted for 89.8% (33/37), C. metapsilosis was only 10.2%. 60% (12/20). The phylogenetic tree based on the ITS region showed that the evolution of C. parapsilosis sensu stricto is more conservative; C.metapsilosis and C. orthopsilosis species are more closely related; C. metapsilosis exists two types of ITS genotypes (I and II), mainly in type II. Conclusion (1) the near smooth Candida complex is in China There are regional differences in the distribution of species in East China. (2) there are different ITS genotypes in C. metapsilosis strains.
The second chapter is PCR-RFLP and RAPD identification typing of Candida albicans complex.
Objective to construct and evaluate the PCR-RFLP and RAPD-PCR methods for molecular identification of Candida albicans, and to further RAPD fingerprint classification and cluster analysis of clinical strains. Method (1) POM-StuI/StyI enzyme digestion: Design interspecific polymorphic fragment POM, PCR amplification of ITS region, POM and PCR in ITS region. The fragment was reacted with StuI and StyI respectively. (2) random primer RP02 was used to amplify the Genomic Polymorphic fragment of the clinical strain by RAPD-PCR. The RAPD fingerprint was formed and the RAPD fingerprint was clustered. The results were (1) POMf/r specific amplification of C. parapsilosis sensu stricto. The POM fragment of the region, after StuI and StyI enzyme digestion, formed a specific RFLP band, which was consistent with the sequencing identification results. (2) RP02-RAPD reaction produced C. parapsilosis sensu stricto and C. metapsilosis RAPD fingerprint. Conclusion (1) POM-StuI/StyI enzyme digestion method can achieve rapid and accurate molecular identification of Candida albicans complex; (2) RPO2-RAPD can also realize C. parapsilosis sensu stricto and C. metapsilosis rapid molecular identification; (3)'C. metapsilosis has 2 differential distribution of RAPD genotypes, and the same as ITS genotypes.
The third chapter is about drug sensitivity of Candida albicans complex.
Objective to determine the sensitivity of the Candida albicans complex to common antifungal agents. Methods the sensitivity of the Candida albicans complex clinical strain to amphotericin B, fluconazole, itraconazole, voriconazole and Mika Finn Jing was tested by the American CLSIM27-A3 guide. Results all strains were susceptible to amphotericin B, fluconazole, and volamoconazole. Raconazole and Mikafin were all sensitive, 6 C. parapsilosis sensu stricto and 3 strains of C.metapsilosis were sensitive to itraconazole. No drug resistant strains were found. Conclusion the drug resistant strains of Candida albicans were rare, and common antifungal drugs could effectively treat Candida albicans infection.
The fourth chapter is extracellular hydrolase from Candida albicans complex.
Objective to detect the ability of 34 strains of Candida albicans (21 strains of C. parapsilosis sensu stricto, 1 C. orthopsilosis and 12 C. metapsilosis) to produce extracellular hydrolase. Methods using the yolk medium, fetal bovine albumin culture and Twain -80 medium to detect phospholipase and eggs of the Candida albicans compound strain respectively. The ability of white enzyme and esterase. Results (1) phospholipase activity: 90.5% (19/21) C. parapsilosis sensu stricto and 91.7% (11/12) C. metapsilosis strains produce phospholipase activity, and there is no significant difference in the activity of two fungal enzymes. C. orthopsilosis standard strain does not detect phospholipase activity; (2) protease activity: 81% (17/21) C. Su stricto and 83.3% (10/12) C. metapsilosis strain expressed protease activity, the former was more active; C. orthopsilosis standard strain expressed the extremely strong protease activity; (3) esterase activity: only 4.76%C. parapsilosis sensu stricto (1/21) and esterase activity were expressed. Conclusion in this study, C.parapsilosis sensu stricto and C. metapsilosis are similar to Candida albicans, both of which express phospholipase and protease activity, but the activity of esterase is very low.
The second part is the identification method of Candida in Dublin.
The fifth chapter is the classical phenotypic method and molecular biological identification of Candida albicans in Dublin.
Objective to compare the classical phenotypic identification methods and construction of 6 species of Candida albicans in Dublin, and to evaluate a new method for identification of colony PCR-RFLP molecules (1) using Colma coloured culture medium, the formation test of thick wall spores of corn flour Twain medium, tolerance test at 45 C, tolerance test of hypertonic culture base, morphological observation of tobacco leaf culture medium and API20C AUX birth. 6 classical phenotypic methods, such as chemical identification system, were identified for Candida albicans and Dublin candidal standard strains and 66 Candida albicans / Candida albicans; (2) colony PCR-ApaI/BsiEI enzyme digestion: colony PCR amplification of Candida albicans, Candida albicans in Dublin and other 8 common Candida standard strains of D1-D2, ApaI and BsiEI No enzymatic identification; and molecular identification of clinical isolates. Results (1) high permeability medium tolerance test, tobacco leaf culture basis morphological observation can accurately identify Candida albicans and Dublin candidal standard strain; 66 clinical strains are Candida albicans by phenotypic identification; (2) colony PCR-ApaI/BsiEI producing Candida albicans and Dublin Candida specific RFLP banding pattern. 66 clinical isolates were all Candida albicans by molecular identification.
Conclusion (1) the combination of 2 or more phenotypic identification methods can be used to identify and identify Candida albicans in Dublin. (2) colonies PCR-ApaI/BsiEI can be used to identify fast and accurate molecular identification of Candida albicans and Candida albicans in Dublin.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R379

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