眼部感染热带念珠菌基因分型的初步研究  

A preliminary study on genotype of candida tropicalis isolated from eyes infection

作　　者：宋绪华[1] 邢健强[1] 陈海[1] 陈梨萍[1] 

机构地区：[1]海南医学院附属医院眼科,海口570102

出　　处：《眼科研究》2010年第12期1115-1118,共4页Chinese Ophthalmic Research

基　　金：海南省自然科学基金项目(807094)

摘　　要：目的建立随机扩增DNA多态性(RAPD)反应体系对热带念珠菌进行基因分型。方法从14例真菌感染的眼部病变处取材并根据真菌菌落外观、菌丝、孢子及菌细胞形态进行菌种鉴定,采用Neo-Sensitab抗真菌药敏纸片进行药物敏感试验。分别提取14株眼部感染热带念珠菌临床分离株的基因组DNA,依次用随机引物OPA-18、OPE-18、RSD-6、OPG-10对其进行PCR扩增,图谱分析后用非加权配对算术平均法(UPGMA)绘出亲缘关系聚类图。结果 14株热带念珠菌对两性霉素B、酮康唑、氟康唑和氟胞嘧啶的敏感率依次为50.00%、85.71%、100.00%和100.00%。采用引物OPG-10和RSD-6构建的RAPD-PCR体系可以扩增出质量较好的带谱,多态性位点依次为71.42%和72.41%。采用引物OPG-10进行RAPD-PCR产物构建的亲缘关系聚类图中,对两性霉素B敏感的第1、2、3、4、5、6株热带念珠菌与对两性霉素B耐药的第14株热带念珠菌聚成一簇,而其余对两性霉素B耐药的第7、8、9、10、11、12、13株聚成一簇。采用引物RSD-6进行RAPD-PCR产物构建的亲缘关系聚类图中,对两性霉素B敏感的第1、2、3、4、5、6株聚成一簇,而对两性霉素B耐药的第7、8、9、10、11、12、13、14株聚成一簇。结论采用引物RSD-6构建的RAPD-PCR反应体系可以较好地进行热带念珠菌的分子分型,对两性霉素B敏感株和耐药株进行鉴定。Background Candida tropicalis infection has been listed in the second in fungi infection and first in non-Candida albicans infection.Candida tropicalis can result in invasive infection,hematologic tumors,endocarditis.Therefore,it is very important to study the typing and biology behavior of candida tropicalis.ObjectiveThis study attempted to develop the random amplified polymorphic DNA（RAPD）system for the genotyping of candida tropicalis isolated from infected eye.MethodsSpecimens were taken from eye lesions in 14 cases with fungi infection,and submitted for fungi identification according to its appearance,mycelium,spores and bacteria cells morphology.Then drug sensitivity test were conducted with Neo-Sensitab antifungal susceptibility paper provided by ROSCO Company of Denmark.The genomic DNA was extracted from 14 clinical stains of candida tropicalis,and amplified by PCR with random primers of OPA-18,OPE-18,RSD-6 and OPG-10 respectively.The RAPD-PCR products were analyzed and mapped for the relationship dendrogram with unweighted pair grouping method with arithmetic mean（UPGMA）.ResultsThe sensitivity rates of the 14 clinical strains of candida tropicalis were 50.00% to amphotericin B,85.71% to ketoconazole,100.00% to fluconazole,and 100.00% to flucytosine.The maps with good quality were obtained with RAPD-PCR systems using random primers of OPG-10 and RSD-6,and their polymorphic sites were 71.42% and 72.41%.According to the relationship dendrogram mapped with random primers of OPG-10,the stains of 1,2,3,4,5,6 sensitive to amphotericin were clustered with the strain 14 resistant to amphotericin,and the other 7,8,9,10,11,12 and 13 strains resistant to amphotericin were clustered.At the same time,the stains of 1,2,3,4,5 and 6 sensitive to amphotericin were clustered,but the strains of 7,8,9,10,11,12,13 and 14 were clustered.ConclusionThe RAPD-PCR system constructed with random primers of RSD-6 can classify candida tropicalis into sensitive and resistant strains at the molecular level.

关 键 词：热带念珠菌 随机扩增DNA多态性 基因分型 两性霉素B 药物敏感试验 

分 类 号：R771.3[医药卫生—眼科]