本文選題：隱球菌　切入點：新型　出處：《第二軍醫(yī)大學》2008年碩士論文

【摘要】： 目的調(diào)查1993.3-2007.12分離自我國東南地區(qū)115株新生臨床株的基因型組成、配型組成、地域分布等進行分析,為我國隱球菌病的預(yù)防、診療提供科學的依據(jù)。 方法1.PCR指紋分型:以野生噬菌體M13的小衛(wèi)星核心序列為單引物對模板DNA進行PCR擴增,參照標準菌株將臨床株鑒定至8種基因型;2.多位點基因序列及系統(tǒng)進化學分析:對115株新生隱球菌臨床株的ITS和CAP59基因進行PCR擴增、測序、序列分析;CLUSTALW1.83軟件多重比對分析ITS序列的差別,MEGA3.1軟件處理數(shù)據(jù)NJ法繪制系統(tǒng)進化樹,bootstrapping法對系統(tǒng)進化樹結(jié)果進行統(tǒng)計學檢驗,區(qū)分新生隱球菌grubii變種、neoformans變種及gattii變種菌株;對不同血清型隱球菌菌株的CAP59基因序列進行比對及系統(tǒng)發(fā)育學分析,探索區(qū)分新生隱球菌血清型的可行性;利用MLST技術(shù),分析S8012與國外格特菌株的遺傳進化關(guān)系;3.根據(jù)Chaturvedi et al等描述的方法選用特異性引物PCR特異性擴增相關(guān)基因:鑒定α和a配型。 結(jié)果1.分離自東南地區(qū)115株隱球菌臨床株中,88株(76.5%)為VNⅠ基因型菌株;17株(14.8%)為VNⅡ基因型菌株;3株(2.6%)為VNⅢ基因型菌株;1株(0.87%)為VNⅣ基因型菌株;5株(4.3%)為VGⅠ基因型;1株(0.87%)為VGⅡ基因型;2.ITS序列分析可以明確的將各臨床株鑒定至變種水平;CAP59部分基因序列分析不能明確鑒定新生隱球菌血清型;3.國內(nèi)115株隱球菌臨床分離株112株為α配型菌株,占97.4%;1株為α/a配型菌株;2株為α/-配型菌株;4.分離到5株2種基因型新生隱球菌格特變種菌臨床株,見于上海、江蘇南部、浙江北部及廣東省地區(qū)。 結(jié)論1.分離自中國東南地區(qū)的新生隱球菌臨床株以α配型,VNⅠ基因型菌株為主;分離自該地區(qū)AIDS患者的臨床株亦以α,VNⅠ基因型菌株為主,但也有α/a,VNⅢ基因型菌株;2.中國新生隱球菌臨床株存在一定的遺傳多態(tài)性,其中格特變種臨床株主要分離自上海、江蘇南部、浙江北部和廣東等地區(qū);3.中國新生隱球菌臨床株配型中α配型占絕大部分;4.ITS結(jié)合CAP59基因序列及系統(tǒng)發(fā)育學分析可將新生隱球菌鑒定至變種水平。
[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，

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