裴氏着色霉的随机扩增DNA多态性研究

Characterization of Fonsecaea Pedrosoi by Random Amplification of Polymorphic DNA Assay

目的 进一步了解裴氏着色霉的基因学特征 ,采用溴苯提取法提取基因组DNA。方法 以随机扩增DNA多态性法对来源于 7个国家的 3 2株裴氏着色霉 (日本 15株 ,中国 3株 ,委内瑞拉 4株 ,中美洲 4株 ,哥伦比亚 2株 ,墨西哥 2株 ,巴西 1株 ,泰国 1株 )的DNA指纹图谱作了分析。结果 ①共选用 10个随机引物进行扩增 ,筛选出 3个具有较好DNA扩增片段的引物 ,即引物 1:5’ GAGCC CTCCA 3’ ,引物 7:5’ CGGCCCCTGT 3’ ,引物 8:5’ AGGTCACTGA 3’。② 3 2株裴氏着色霉的DNA带型不完全相同 ,具有一定的遗传变异性。③来源于同一国家的菌株具有较高的遗传相似性。④来源于日本的 15株菌株采用引物 8扩增后呈现两种DNA带型 ,I型 (有 3条主带 ) ,Ⅱ型 (有两条主带 ) ,其余国家菌株由于数量较少 ,DNA分型不明显。⑤同一引物扩增 ,90 %以上菌株可获共同的DNA片段。结论 随机扩增DNA多态性研究发现裴氏着色霉具有一定的种内差异 ,其DNA带型与菌株的来源地域关系密切 ,本研究方法简便快速 ,可用于着色芽生菌病的分子流行病学调查及可能的基因学诊断中。

Objective To investigate the DNA polymorphism of F pedrosoi and to find the relationship between DNA patterns and morphology in different geographic areas Methods The total DNA was extracted with benzyl bromide Random amplification of polymorphic DNA(RAPD) assay was used to study the genetic similarity (GS) of 32 isolates from seven countries (15 isolates from Japan, 3 isolates from China, 4 from Venezuela, 4 from Central America, 2 from Columbia, 2 from Mexico, 1 from Brazil and 1 from Thailand ) Results ①Of ten random primers used, three (primers 1,7,8) gave good reactions The sequences of them were: primer 1: 5′ GAGCCCTCCA 3′, primer 7: 5′ CGGCCCCTGT 3′, primer 8: 5′ AGGTCACTGA 3′, ②The RAPD patterns of 32 isolates showed some degrees of variation ③The isolates from the same country showed high genetic similarities ④15 isolates from Japan could be divided into 2 types by RAPD analysis with primer 8 ⑤With the same primer,the common DNA fragment could be gotten from more than 90%isolates Conclusion There are some genetic variations between 32 isolates of F pedrosoi The genetic similarity of isolates is closely related to the geographic origin As a simple and rapid method, RAPD assay can be used for the study of epidemiology and genetic characterization of chromoblastomycosis