PCR-RFLP和PCR指纹法在新生隐球菌基因分型中的应用与比较  

PCR-RFLP versus PCR fingerprinting method in the genotyping of Cryptoeoeeus neoformans

作　　者：冯晓博[1] 姚志荣[1] 李晓辉[2] 任大明[2] 

机构地区：[1]上海交通大学医学院附属新华医院皮肤科,200092 [2]复旦大学遗传所遗传工程国家重点实验室

出　　处：《中华皮肤科杂志》2008年第4期226-229,共4页Chinese Journal of Dermatology

摘　　要：目的探讨针对结构基因g6341的PCR-RFLP方法在新生隐球菌基因分型中的应用价值，并与PCR指纹分型法进行比较。方法以野生型噬菌体M13中针对小卫星DNA的核心序列为单引物，对受试的新生隐球菌进行PCR指纹分型。选择结构基因g6341进行PCR-RFLP分析，在序列保守区设计通用引物，筛选合适的限制性内切酶进行RFLP分析，并与PCR指纹法进行比较。g6341基因扩增片段测序，进行系统发育分析，研究各主要基因型间的亲缘关系。结果多位点基因序列分析表明，结构基因g6341可作为RFLP分析的合适靶点。对76株新生隐球菌的基因分型结果显示，针对g6341的PCR-RFLP方法与PCR指纹法结果一致。分析g6341基因部分序列得出，8种主要基因型菌株相互间的同源性在84％-97％之间，同一主要基因型菌株的同源性在99％-100％之间。分子发育树中，VNⅠ—VNⅣ基因型、VGⅠ—VGⅣ基因型分别聚在一类。结论针对结构基因g6341的PCR-RFLP方法可作为新生隐球菌分子分型的有效工具，对g6341基因的序列分析可揭示不同菌株间的遗传进化关系。Objective To evaluate the efficiency of PCR-restriction fragment length polymorphism （PCR-RFLP） aiming at the structure gene g6341, versus PCR fingerprinting analysis in the genotyping of Cryptococcus neoformans. Methods Eight reference strains and 68 clinical and environmental isolates of C. neoformans were genotyped by PCR-RFLP and PCR fingerprinting. In PCR fingerprinting, the minisatel lite-specific core sequence of wild-type phage M13 was used as a single primer. The structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes, a pair of primers were developed based on the conserved region of g6341 gene. PCR products were digested with the appropriate restriction endonucleases, and RFLP profiles were analyzed. Partial sequence analysis of g6341 gene was performed for different genotypes of C. neoformans. Phylogenetic analysis was done to study the relatedness between these genotypes. Resttlts As sequence homology analysis showed, g6341 gene was suitable for RFLP analysis. In the case of genotyping of 76 C. neoformans strains, the results obtained from PCR-RFLP were consistent with those from PCR fingerprinting. Sequence analysis of g6341 gene revealed a homology of 84%-97% among the eight genotypes as well as a consistency of 99%-100% within a same genotype. In the phylogenetic tree, genotypes VN Ⅰ, VNⅡ, VNⅢ and VNⅣ belonged to one cluster, and genotypes VG Ⅰ, VG Ⅱ, VGⅢ and VGⅣ to another cluster. Conclusions PCR-RFLP analysis aiming at the structure gene g6341 is a useful tool to genotype C. neoformans. Sequence analysis of g6341 gene can disclose the relatedness among different molecular types of C. neoformans.

关 键 词：隐球菌 新型 基因型 DNA指纹法 序列分析 DNA 

分 类 号：R450[医药卫生—治疗学]