利用核糖体基因进行孢子丝菌种内分型及一株播散型孢子丝菌的基因鉴定

发布时间：2018-06-17 02:49

  本文选题：申克氏孢子丝菌 + 孢子丝菌病　； 参考：《大连医科大学》2005年硕士论文

【摘要】：申克氏孢子丝菌是孢子丝菌病的致病菌,主要引起皮肤、皮下组织和附近淋巴系统的亚急性和慢性感染,偶可播散至骨骼和内脏,甚至发生全身播散性感染。真菌传统的分型方法主要依赖菌体培养和镜下的形态学观察,而申克氏孢子丝菌具有“表型特征基本一致”的特点,此时,利用分子生物学方法对其进行基因分型就显示出一定的优势。本研究采用核糖体基因限制性内切酶多态性分析(rDNA RFLP)与Southern blotting 杂交相结合的方法对孢子丝菌进行种内分型, 探讨孢子丝菌的基因学特征,研究基因分型与菌种地区来源及临床表现的关系。同时应用常规真菌学和PCR-序列分析方法对其中一株致皮肤播散型孢子丝菌病的临床分离株(Dmu11)进行鉴定,并探讨该菌株与皮肤淋巴管型、固定型孢子丝菌菌株在基因水平上的异同。 材料和方法:1.31 株来源于不同地区及不同临床类型的申克氏孢子丝菌临床分离株。CTAB 法提取菌体基因组DNA;以真菌通用引物ITS4[5‘TCCTC CGCTT ATTGA TATGC3’] 、NS5[5‘AACTT AAAGG AATTG ACGGA AG 3’]扩增ATCC10268 的rDNA 序列作为探针,与经限制性内切酶ApaI完全酶切后的31 株孢子丝菌DNA 进行印记杂交。以杂交带型作为对孢子丝菌进行基因分型的依据。2. 常规真菌学鉴定和使用PCR-序列分析方法扩增一株致皮肤播散型孢子丝菌病临床分离株(Dmu11)的核糖体保守区(5.8SrDNA,ITSⅡ)基因、非转录区(NTS)基
[Abstract]:Sporocystis schenckii is a pathogen of spore filariasis, which mainly causes subacute and chronic infections in skin, subcutaneous tissue and adjacent lymphatic system, occasionally spreading to bone and viscera, and even to systemic disseminated infection. The traditional classification methods of fungi mainly depend on cell culture and morphological observation under microscope, while Sporocystis schenckii has the characteristics of "basically consistent phenotypic characteristics", at this time, Using molecular biological methods to genotyping them shows certain advantages. In this study, ribosomal restriction endonuclease polymorphism analysis and Southern blotting hybridization were used to study the genetic characteristics of spore filaments. To study the relationship between genotyping and the origin and clinical manifestations of bacteria. At the same time, routine mycology and PCR- sequence analysis were used to identify one of the clinical isolates of dermatogenic sporocystis, Dmu11, and to explore the relationship between the strain and the lymphatic type of skin. The differences and similarities in the gene level of the strains of Sporocystis immobilized. Materials and methods Genomic DNA was extracted from clinical isolates of Sporocystis schenckii from different regions and different clinical types. CTAB method was used to extract genomic DNA, and NS5 [5AACTT AAAGG AATTG ACGGA AG3'] was expanded with the universal primer ITS4 (TCCTC CGCTT ATTGA TATGC3'). The rDNA sequence of ATCC10268 was used as a probe. The DNA of 31 strains of spore filaments digested by restriction endonuclease ApaI was hybridized with imprinting. The hybridization band type was used as the basis for genotyping of spore filaments. Routine mycological identification and PCR- sequence analysis to amplify the ribosomal conserved region of its 鈪，

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