白念珠菌多位点序列分型研究
发布时间：2018-11-24 11:52
【摘要】：目的应用多位点序列分型(Multilocus Sequence Typing, MLST)对临床分离白念珠菌进行分型研究,同时与ABC (ATP-binding cassette)分型、随机扩增多态DNA技术(randomLy amplified polymorphic DNA, RAPD)结果比较,研究菌株分型与标本来源、药物敏感性等之间的关系,建立国内白念珠菌MLST数据库。 方法 1.收集临床分离白念珠菌,进行体外药物敏感性测定,选取10株氟康唑耐药株,23株氟康唑敏感株,提取菌株DNA。 2.建立MLST分型方法,选择7对管家基因进行PCR扩增,并将目的基因产物测序,然后将测序结果与标准序列比对后上传至MLST网站数据库,获得相应的由7对管家基因组成的等位基因谱,最后将等位基因谱再次提交网站确认序列型(Sequence types, STs). 3.选取ABC分型和RAPD特定引物进行PCR扩增,产物经琼脂糖凝胶电泳后确定分型。 4.将MLST结果经MEGA4.O生物信息学分析软件分析后,采用非加权分组算术均方根法(UPGMA)制作最小生成树,进一步分析ST与菌株信息、其他分型方法的关系。 结果 1.33株临床白念珠菌通过MLST产生了33个STs,采用ABC方法分类为3型,采用RAPD方法引物RSD-12产生13型,引物OPE-18产生11型。 2.最小生成树选取区分值0.0025作为分界线划定组,产生8个组,显示白念珠菌的MLST型别呈现多样性,广泛分布在各个进化支上。 结论 1. MLST具有较高的分辨力,是一种方便、快速的分子生物学方法,可用于流行病学和菌群多态性的研究。 2.聚类分析表明MLST分型结果与ABC分型有关系,但与菌株的标本采集来源、医院来源、RAPD分型结果或抗真菌药耐药性无明显相关。 3.初步建立了MLST分型方法和国内白念珠菌MLST数据库。
[Abstract]:Objective to study the clinical isolates of Candida albicans by multilocus sequence typing (Multilocus Sequence Typing, MLST), and to compare the results of (randomLy amplified polymorphic DNA, RAPD) with ABC (ATP-binding cassette) typing and random amplified polymorphic DNA (DNA). The MLST database of Candida albicans was established by studying the relationship between strain typing and sample source, drug sensitivity and so on. Method 1. Clinical isolates of Candida albicans were collected and tested for drug sensitivity in vitro. Ten fluconazole-resistant strains and 23 fluconazole-sensitive strains were selected to extract DNA.. 2. MLST typing method was established, 7 pairs of housekeeping genes were selected for PCR amplification, and the target gene products were sequenced. The results were compared with standard sequences and uploaded to the MLST website database. The corresponding allelic spectrum consisting of 7 pairs of housekeeper genes was obtained. Finally, the allele spectrum was resubmitted to the website to confirm the sequence of (Sequence types, STs). 3. ABC typing and RAPD specific primers were selected for PCR amplification and the products were identified by agarose gel electrophoresis. 4. The MLST results were analyzed by MEGA4.O bioinformatics analysis software, and the minimal spanning tree was made by (UPGMA) with unweighted grouping arithmetic RMS method. The relationship between ST and strain information and other typing methods was further analyzed. Results 1. 33 strains of clinical Candida albicans produced 33 STs, types by ABC, 13 types by RAPD primer RSD-12 and 11 types by primer OPE-18. 2. 2. The minimum spanning tree selected 0.0025 as the dividing line and produced eight groups, which showed that the MLST types of Candida albicans showed diversity and were widely distributed in various evolutionary branches. Conclusion 1. MLST has high resolution and is a convenient and rapid molecular biological method, which can be used in epidemiology and microbial polymorphism research. 2. Cluster analysis showed that MLST typing was related to ABC typing, but not to sample collection, hospital origin, RAPD typing or antifungal drug resistance. 3. MLST typing method and MLST database of Candida albicans were established.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
本文编号：2353601
[Abstract]:Objective to study the clinical isolates of Candida albicans by multilocus sequence typing (Multilocus Sequence Typing, MLST), and to compare the results of (randomLy amplified polymorphic DNA, RAPD) with ABC (ATP-binding cassette) typing and random amplified polymorphic DNA (DNA). The MLST database of Candida albicans was established by studying the relationship between strain typing and sample source, drug sensitivity and so on. Method 1. Clinical isolates of Candida albicans were collected and tested for drug sensitivity in vitro. Ten fluconazole-resistant strains and 23 fluconazole-sensitive strains were selected to extract DNA.. 2. MLST typing method was established, 7 pairs of housekeeping genes were selected for PCR amplification, and the target gene products were sequenced. The results were compared with standard sequences and uploaded to the MLST website database. The corresponding allelic spectrum consisting of 7 pairs of housekeeper genes was obtained. Finally, the allele spectrum was resubmitted to the website to confirm the sequence of (Sequence types, STs). 3. ABC typing and RAPD specific primers were selected for PCR amplification and the products were identified by agarose gel electrophoresis. 4. The MLST results were analyzed by MEGA4.O bioinformatics analysis software, and the minimal spanning tree was made by (UPGMA) with unweighted grouping arithmetic RMS method. The relationship between ST and strain information and other typing methods was further analyzed. Results 1. 33 strains of clinical Candida albicans produced 33 STs, types by ABC, 13 types by RAPD primer RSD-12 and 11 types by primer OPE-18. 2. 2. The minimum spanning tree selected 0.0025 as the dividing line and produced eight groups, which showed that the MLST types of Candida albicans showed diversity and were widely distributed in various evolutionary branches. Conclusion 1. MLST has high resolution and is a convenient and rapid molecular biological method, which can be used in epidemiology and microbial polymorphism research. 2. Cluster analysis showed that MLST typing was related to ABC typing, but not to sample collection, hospital origin, RAPD typing or antifungal drug resistance. 3. MLST typing method and MLST database of Candida albicans were established.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
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