近平滑念珠菌和都柏林念珠菌的表型和基因型研究

发布时间：2018-06-05 05:19

本文选题：近平滑念珠菌复合体 + 都柏林念珠菌　；参考：《北京协和医学院》2011年博士论文

【摘要】：第一部分近平滑念珠菌复合体表型与基因型研究第一章近平滑念珠菌复合体的筛选、测序鉴定和种系进化分析目的研究我国华东地区近平滑念珠菌复合体的菌种分布和种系进化关系。方法利用科玛嘉念珠菌显色培养基和API20CAUX生化系统对分离白华东地区4个城市的80株临床株进行表型鉴定。经生化鉴定为近平滑念珠菌、季也蒙念珠菌、无名念珠菌和葡萄牙念珠菌的菌株进一步进行ITS区测序分子鉴定,构建基于ITS区序列的种系进化树,分析近平滑念珠菌复合体中菌株的种系亲缘关系。结果80株临床株中,60株菌株(75%)经生化系统鉴定为近平滑念珠菌,11株(13.75%)为光滑念珠菌,4株(5%)为季也蒙念珠菌,葡萄牙念珠菌、乳酒念珠菌、涎沫念珠菌、郎比卡念珠菌和无名念珠菌各1株(1.25%)。60株表型鉴定为近平滑念珠菌中,3株分子鉴定为季也蒙念珠菌,57株菌为近平滑念珠菌复合体,其中Candida parapsilosis sensu stricto占71.9%(41/57),C. metapsilosis占28.1%(16/57)；未分离到Corthopsilosis。菌种分布呈现显著的地域差异：来自南京、上海和济南的37株临床株中,C. parapsilosis sensu stricto占89.8%(33/37),C. metapsilosis仅占10.2%(4/37)；而来源于南昌的临床株中则以C. metapsilosis为主,占60%(12/20)。基于ITS区的种系进化树表明：C. parapsilosis sensu stricto进化上较为保守；C.metapsilosis和C. orthopsilosis种系亲缘关系更为紧密；C. metapsilosis存在两种分布不均的ITS基因型(Ⅰ和Ⅱ),以Ⅱ型为主。结论(1)近平滑念珠菌复合体在我国华东地区的菌种分布存在地区差异；(2) C. metapsilosis菌株中存在差异分布的ITS基因型。第二章近平滑念珠菌复合体的PCR-RFLP和RAPD鉴定分型研究目的构建和评价用于近平滑念珠菌复合体分子鉴定的PCR-RFLP和RAPD-PCR方法,并进一步对临床株进行RAPD指纹分型和聚类分析。方法(1)POM-StuI/StyI酶切法：设计近平滑念珠菌复合体特异性引物POMf/r扩增ITS区的种间多态性片段POM, PCR扩增POM片段,后分别用StuI和StyI进行酶切反应。(2)随机引物RP02进行RAPD-PCR扩增临床株基因组多态性片段,形成RAPD指纹,并对RAPD指纹进行聚类分析。结果(1) POMf/r特异性扩增C. parapsilosis sensu stricto, C. orthopsilosis和C. metapsilosis标准株和临床株的ITS区POM片段,经StuI和StyI酶切后形成种特异性RFLP条带,与测序鉴定结果一致。(2) RP02-RAPD反应产生C. parapsilosis sensu stricto和C. metapsilosis种特异性RAPD指纹图谱；RAPD指纹聚类分析表明：C. metapsilosis存在2种差异分布的基因型,与ITS基因型相一致。结论(1) POM-StuI/StyI酶切法可以实现近平滑念珠菌复合体的快速而准确的分子鉴定；(2)RPO2-RAPD也可以实现C. parapsilosis sensu stricto和C. metapsilosis快速分子鉴定；(3)'C. metapsilosis存在2种差异分布的RAPD基因型,与ITS基因型一致。第三章近平滑念珠菌复合体药物敏感性研究目的测定近平滑念珠菌复合体对常见抗真菌药物的敏感性。方法参照美国CLSIM27-A3指南进行药敏试验,检测近平滑念珠菌复合体临床株对两性霉素B、氟康唑、伊曲康唑、伏立康唑和米卡芬净的敏感性。结果所有菌株对两性霉素B、氟康唑、伏立康唑和米卡芬净均敏感,6株C. parapsilosis sensu stricto和3株C.metapsilosis对伊曲康唑呈剂量依赖性敏感。未发现耐药株。结论临床上近平滑念珠菌耐药株罕见,常见抗真菌药物可以有效治疗近平滑念珠菌感染。第四章近平滑念珠菌复合体胞外水解酶研究目的检测34株近平滑念珠菌复合体菌株(21株C. parapsilosis sensu stricto,1株C. orthopsilosis和12株C. metapsilosis)产生胞外水解酶的能力。方法利用卵黄培养基、胎牛白蛋白培养基和吐温-80培养基分别检测近平滑念珠菌复合体菌株产生磷脂酶、蛋白酶和酯酶的能力。结果(1)磷脂酶活力：90.5%(19/21)C. parapsilosis sensu stricto和91.7%(11/12)C. metapsilosis菌株产生磷脂酶活力,两种真菌酶活力无显著性差异,C. orthopsilosis标准株未检测到磷脂酶活力；(2)蛋白酶活力：81.0%(17/21) C. parapsilosis sensu stricto和83.3%(10/12)C. metapsilosis菌株表达蛋白酶活力,前者活力更强；C. orthopsilosis标准株表达极强水平的蛋白酶活力；(3)酯酶活力：仅有4.76%C. parapsilosis sensu stricto (1/21)和16.7%C.metapsilosis (2/12)表达酯酶活力,C. orthopsilosis标准株未表达酯酶活力。结论本研究中C.parapsilosis sensu stricto, C. metapsilosis与白念珠菌类似,均高表达磷脂酶和蛋白酶活力,但酯酶活力极低。第二部分都柏林念珠菌的鉴定方法研究第五章都柏林念珠菌的经典表型方法与分子生物学鉴定方法研究目的比较6种都柏林念珠菌经典表型鉴定方法和构建、评价一种新的菌落PCR-RFLP分子鉴定方法方法(1)利用科玛嘉显色培养基、玉米粉吐温培养基厚壁孢子形成试验、45℃耐受试验、高渗培养基耐受试验、烟叶培养基形态学观察和API20C AUX生化鉴定系统等6种经典表型方法鉴定白念珠菌和都柏林念珠菌标准株和66株白念珠菌/都柏林念珠菌临床分离株；(2)菌落PCR-ApaI/BsiEI酶切法：菌落PCR扩增白念珠菌、都柏林念珠菌和其它8种常见致病酵母菌标准株的D1-D2区,用ApaI和BsiEI分别进行酶切鉴定；并对临床分离株进行分子鉴定。结果(1)高渗培养基耐受试验、烟叶培养基形态学观察可以准确鉴定白念珠菌和都柏林念珠菌标准株；66株临床株经表型鉴定均为白念珠菌；(2)菌落PCR-ApaI/BsiEI产生白念珠菌和都柏林念珠菌特异性RFLP带型,66株临床株经分子鉴定均为白念珠菌。结论(1)联合2种或更多种表型鉴定方法可以实现都柏林念珠菌的筛选和初步鉴定；(2)菌落PCR-ApaI/BsiEI可以实现白念珠菌和都柏林念珠菌的快速准确的分子鉴定。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R379

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