中国东南地区115株新生隐球菌临床株配型和分子流行病学研究

发布时间：2018-03-26 04:39

  本文选题：隐球菌　切入点：新型　出处：《第二军医大学》2008年硕士论文

【摘要】： 目的调查1993.3-2007.12分离自我国东南地区115株新生临床株的基因型组成、配型组成、地域分布等进行分析,为我国隐球菌病的预防、诊疗提供科学的依据。 方法1.PCR指纹分型:以野生噬菌体M13的小卫星核心序列为单引物对模板DNA进行PCR扩增,参照标准菌株将临床株鉴定至8种基因型;2.多位点基因序列及系统进化学分析:对115株新生隐球菌临床株的ITS和CAP59基因进行PCR扩增、测序、序列分析;CLUSTALW1.83软件多重比对分析ITS序列的差别,MEGA3.1软件处理数据NJ法绘制系统进化树,bootstrapping法对系统进化树结果进行统计学检验,区分新生隐球菌grubii变种、neoformans变种及gattii变种菌株;对不同血清型隐球菌菌株的CAP59基因序列进行比对及系统发育学分析,探索区分新生隐球菌血清型的可行性;利用MLST技术,分析S8012与国外格特菌株的遗传进化关系;3.根据Chaturvedi et al等描述的方法选用特异性引物PCR特异性扩增相关基因:鉴定α和a配型。 结果1.分离自东南地区115株隐球菌临床株中,88株(76.5%)为VNⅠ基因型菌株;17株(14.8%)为VNⅡ基因型菌株;3株(2.6%)为VNⅢ基因型菌株;1株(0.87%)为VNⅣ基因型菌株;5株(4.3%)为VGⅠ基因型;1株(0.87%)为VGⅡ基因型;2.ITS序列分析可以明确的将各临床株鉴定至变种水平;CAP59部分基因序列分析不能明确鉴定新生隐球菌血清型;3.国内115株隐球菌临床分离株112株为α配型菌株,占97.4%;1株为α/a配型菌株;2株为α/-配型菌株;4.分离到5株2种基因型新生隐球菌格特变种菌临床株,见于上海、江苏南部、浙江北部及广东省地区。 结论1.分离自中国东南地区的新生隐球菌临床株以α配型,VNⅠ基因型菌株为主;分离自该地区AIDS患者的临床株亦以α,VNⅠ基因型菌株为主,但也有α/a,VNⅢ基因型菌株;2.中国新生隐球菌临床株存在一定的遗传多态性,其中格特变种临床株主要分离自上海、江苏南部、浙江北部和广东等地区;3.中国新生隐球菌临床株配型中α配型占绝大部分;4.ITS结合CAP59基因序列及系统发育学分析可将新生隐球菌鉴定至变种水平。
[Abstract]:Objective to investigate the genotypic composition, matching composition and regional distribution of 115 new clinical strains isolated from southeast China in order to provide scientific basis for the prevention, diagnosis and treatment of Cryptococcosis in China. Methods 1.PCR fingerprinting: using the small satellite core sequence of wild bacteriophage M13 as a single primer, the template DNA was amplified by PCR. The clinical strains were identified to 8 genotypes with reference to the standard strain. Multilocus gene sequence and phylochemical analysis: the ITS and CAP59 genes of 115 clinical strains of Cryptococcus neoformans were amplified by PCR and sequenced. The difference of ITS sequence analysis by multiple alignment analysis software CLUSTALW1.83. MEGA3.1 software was used to draw the phylogenetic tree by NJ method. The results of phylogenetic tree were tested by bootstrapping method to distinguish Cryptococcus neoformans var. neoformans from gattii variant strains of Cryptococcus neoformans. The CAP59 gene sequence of Cryptococcus neoformans strains was compared and phylogenetic analysis was carried out to explore the feasibility of distinguishing Cryptococcus neoformans serotype, and MLST technique was used to identify Cryptococcus neoformans serotype. The genetic and evolutionary relationship between S8012 and foreign Gert strain was analyzed. According to the method described by Chaturvedi et al, specific primer PCR was used to amplify the related genes. Results 1. From 115 clinical strains of Cryptococcus in Southeast China, 88 strains of Cryptococcus sp. (76. 5) were identified as VN 鈪，

本文编号：1666369