我国新生隐球菌的基因分型和分子流行病学分析中文摘要本研究通过分子生物学技术，如PCR指纹法、PCR特异扩增、PCR-RFLP、MLST、系统发育研究来分析我国新生隐球菌临床分离株的分子特征，旨在阐明我国新生隐球菌基因型和交配型在临床中的分布及其分子流行病学特征。

主要工作包括以下三个部分：一、PCR-RFLP和PCR指纹法在新生隐球菌基因分型中的应用与比较目的探讨针对结构基因g6341的PCR－RFLP方法在新生隐球菌基因分型中的应用价值, 并与PCR指纹分型法进行比较。

方法以野生型噬菌体M13中针对小卫星DNA的核心序列为单引物，对受试的新生隐球菌进行PCR指纹分型。

选择结构基因g6341进行PCR－RFLP分析，在序列保守区设计通用引物，筛选合适的限制性内切酶进行RFLP分析，并与PCR指纹法进行比较。

g6341基因扩增片段测序，进行系统发育分析，研究各主要基因型间的亲缘关系。

结果多位点基因序列分析表明，结构基因g6341可作为RFLP分析的合适靶点。

对76株新生隐球菌的基因分型结果显示，针对g6341的PCR-RFLP方法与PCR指纹法结果一致。

分析g6341基因部分序列得出，8种主要基因型菌株相互间的同源性在84％～97％之间，同一主要基因型菌株的同源性在99％～100％之间。

分子发育树中，VNI－VNIV基因型、VGI－VGIV基因型分别聚在一类。

结论针对结构基因g6341的PCR-RFLP方法可作为新生隐球菌分子分型的有效工具，对g6341基因的序列分析可揭示不同菌株间的遗传进化关系。

二、国内110株新生隐球菌临床株变种、基因型和交配型分析目的对国内部分地区的新生隐球菌临床株进行分子流行病学调查，分析其变种、基因型和交配型的构成和分布。

方法1、PCR指纹分型法：以野生型噬菌体M13中针对小卫星DNA的核心序列为单引物对模板进行PCR扩增，将所有受试菌株鉴定到8种主要基因型水平。

2、利用变种和交配型特异性引物扩增分型法，区分格鲁比、新生和格特变种，同时鉴定α和a交配型。

结果 110株临床株中，98株（89.1％）为格鲁比变种，均为VNI基因型和α交配型；9株（8.2％）格特变种，包括VGI基因型、α交配型8株（7.3％）和VGII 基因型、α交配型1株（0.9％）；2株（1.8％）为AD杂合体，VNIII 基因型，-/α和α/-交配型各1株；1株（0.9％）为新生变种，VNIV 基因型和a交配型。

结论我国新生隐球菌临床株包含3个变种和AD 杂合体。

与国外情况比较，相似的是国内临床株中绝大部分为α交配型菌株，且格鲁比变种中的VNI基因型占了其中的大部分；但未发现VN II、VG III和VGIV基因型菌株。

三、我国致病格特隐球菌的基因亚型分析目的分析我国致病格特隐球菌的基因型和基因亚型，探讨其与世界范围内格特隐球菌的亲缘关系和分子流行病学联系。

方法扩增国内9株格特隐球菌临床株的3个非连锁位点即IGS1（基因内间隔区）、PLB1（磷脂酶编码基因）和GEF1（交配型位点内基因）部分可变区片段，检索GenBank上相应位点的核酸序列信息，进行多位点的序列比对和系统发育分析；选择针对MF基因(编码性激素) 的PCR特异扩增法鉴定交配型。

结果1、国内8株VGI基因型和α交配型菌株在IGS1、PLB1和GEF1位点上的所测序列与以菌株WM276为代表的一种基因亚型完全相同；国内首株VGII基因型和α交配型菌株在IGS1和PLB1位点所测序列与以菌株R272为代表的一种基因亚型一致。

2、针对GEF1基因部分片段的序列和聚类分析准确鉴定受试格特隐球菌的基因型和交配型。

结论1、多位点序列分析表明，我国致病格特隐球菌VGI基因型与世界上分布较广的一种基因亚型相似，国内首株VGII基因型菌株与温哥华岛致病性弱的VGIIb基因亚型亲缘关系接近。

2、GEF1基因的序列和聚类分析可用于格特隐球菌基因型和交配型的双重鉴定。

关键词：新生隐球菌；基因型；PCR指纹分型；PCR-RFLP；变种；基因型；交配型；格特隐球菌；基因亚型；系统发育分析GENOTYPING AND MOLECULAR EPIDEMIOLOGIC ANALYSIS OF THE CRYPTOCOCCAL ISOLATES FROM CHINAABSTRACTIn this study, several molecular typing techniques, such as PCR fingerprinting, PCR assays with specific primers, PCR-restriction fragment length polymorphism (PCR-RFLP), multilocus sequence typing (MLST) and phylogenetic analysis, were conducted to analyze the population structure and molecular characteristics of the clinical cryptococcal isolates from China. Our aim is to elusidate the geographic distribution of the molecular types and mating types of clinical cryptococcal isolates and the traits of molecular epidemiology of this pathogen in China. The whole work was classified into three parts as follows:Part one:Comparison of PCR-RFLP analysis and PCR fingerprinting in genotyping of Cryptococcus neoformans species complexObjective To evaluate the role of PCR-restriction fragment length polymorphism (PCR-RFLP) aimed at structure gene g6341 in genotypingof Cryptococcus neoformans and compare it with the PCR fingerprinting analysis. Methods Cryptococcus neoformans isolates were subtyped with PCR fingerprinting, in which the minisatellite-specific core sequence of wild-type phage M13 was used as single primer. Structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes. A pair of primers was developed based on conserved region of g6341 gene. PCR products were digested with appropriate restriction endonucleases and RFLP profiles were analyzed. Partial sequence analysis of the gene g6341 of distinct molecular types of Cryptococcus neoformans was done. Phylogenetic analysis was used to study the relatedness between these molecular types. Results Multiple gene sequence analysis showed，g6341 was suitable for RFLP analysis. In subtyping of 76 Cryptococcus neoformans isolates, the results obtained by PCR-RFLP were coincidence with those from PCR fingerprinting. Sequence analysis of g6341 gene showed, strains of eight major molecular types shared 84-97％ homology and strains affiliated to same major molecular type shared 99-100% homology. Two major clusters were apparent in the phylogram, in which cluster 1 included VNI-VNIV molecular types and cluster 2 included VGI-VGIV molecular types. Conclusion PCR-RFLP analysis aimed at structure gene g6341 is a useful tool in genotyping of C ryptococcus neoformans. Sequence analysis of g6341 genes can be used to investigate the relatedness of distinctmolecular types of C ryptococcus neoformans.Part two:Analysis of variety, molecular and mating type of 110 clinical cryptococcal isolates from ChinaObjective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the variety, molecular and mating type (MAT) of them. Methods i) PCR fingerprinting, PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer. Genotypes of the 110 cryptococcal isolates from China were assigned by comparison to the reference strains of the eight major molecular types loaded on gel. ii) Identification of variety and mating type were carried out by PCR using variety and mating type specific primers. Results Of 110 clinical cryptococcal isolates, strains of C. neoformans var. grubii with molecular type VNI and MATαwere the most represented (89.1%), followed by strains of C. neoformans var. gattii (8.2%), including isolates of molecular type VGI, MATα(7.3%) and molecular type VGII, MATα(0.9%); AD hybrids with molecular type VNIII, MAT-/αand molecular type VNIII, MATα/- (1.8%); and isolate of C. neoformans var. neoformans with molecular type VNIV and MATa (0.9%). Conclusion Of the clinical isolates from China, all three varieties and AD hybridswere found. The vast majority (>99%) of strains possessed the α allele in MAT locus and most of them were C. neoformans var. grubii with molecular type VNI, which accorded with most studies of clinical molecular epidemiology in other geographic areas. However, no molecular type VNII, VGIII and VGIV isolates were found in this study.Part three:Molecular analysis of the clinical Cryptococcus gattii isolates from China at subgenotypic levelsObjective Molecular analyses of the clinical Cryptococcus gattii isolates from China at genotypic and subgenotypic levels were conducted to elucidate the relatedness and epidemiological links between the isolates and those from other parts of the world. Methods The partially variable regions of the three unlinked loci, namely IGS1 (the intergenic spacer region), PLB1 (coding phospholipase B) and GEF1 (a gene located at mating type locus), were amplified and sequenced, and the bioinformation in these loci was obtained in GenBank for multilocus sequence alignments and Phylogenetic analyses. The specific amplification of MF gene (coding pheromone) based on PCR reaction was conducted to identify the mating types of Cryptococcus gattii isolates. Results Eight isolates of genotype VGI and mating type α from China had the same allele in all three fragments listed above, respectively, which were all identical with the reference strain WM276, a representativeisolate in a subgenotype. The first isolated genotype VGII and mating type α isolate from China had identical sequence with strain R272 in IGS1 and PLB1 loci. ii) Sequence and Phylogenetic analysis of the fragment of GEF1 gene which located at mating type locus successfully identified the genotypes and mating types of total Cryptococcus gattii isolates in this study. Conclusion i) Multi-locus sequence analyses show that the causative agents of genotype VGI in China are more related to strains from a widely distributed subgenotype in the world, and the first VGII isolate from China resembles a subgenotype VGIIb at Vancouver islands which is less virulent. ii) The Sequence and Phylogenetic analyses of GEF1 gene have the potential value in identification of the genotypes and mating types of Cryptococcus gattii simultaneously.Key words: Cryptococcus neoformans; Molecular type; PCR fingerprinting; PCR-RFLP; Variety; Molecular type; Mating type; Cryptococcus gattii; Subgenotype; Phylogenetic analysis上海交通大学学位论文原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进行研究工作所取得的成果。