Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, India.
Department of Biochemistry, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, India.
J Med Microbiol. 2010 Dec;59(Pt 12):1449-1455. doi: 10.1099/jmm.0.023002-0. Epub 2010 Aug 19.
Rhizopus oryzae is the most frequent causative agent of zygomycosis. Although zygomycosis causes considerable morbidity and mortality in immunocompromised patients, the epidemiology of the disease is not well studied and no standard molecular typing method has been described for any of the causative agents. Here we describe a multilocus microsatellite typing (MLMT) method for R. oryzae. R. oryzae genome sequences were downloaded from the Fungal Genome Initiative database (Broad Institute). The intergenic regions and ORFs of approximately 5.7 Mb were screened for repeat regions with the help of the online repeat search tool Repeat Masker. Of the 30 microsatellite loci identified, 3 microsatellites [RO3, (CCT)(n); RO4, (TA)(n); and RO8, (GAA)(GGA)(n)] were selected after validation of the ability to amplify them and their size variation in 8 randomly selected clinical isolates of R. oryzae. Nucleotide sequence analysis of these loci demonstrated polymorphism in the microsatellite repeat number. The capabilities of these microsatellite loci were assessed for strain differentiation on 30 clinical isolates, based on fragment size determination in an automated capillary electrophoresis using fluorescent labelled primers. These three polymorphic microsatellite loci were found to have good discriminatory power (D) (RO3, D=0.846; RO4, D=0.747; RO8, D=0.742; with a combined D=0.986) and stability for seven subcultures. It was also confirmed that the MLMT method may be applied to both R. oryzae and Rhizopus delemar (a proposed new species), although MLMT analysis could not differentiate them into two clusters. The MLMT system, described here for what is believed to be the first time for a zygomycotic fungus, holds promise as a powerful tool for the strain typing of R. oryzae.
米根霉是最常见的接合菌病病原体。尽管接合菌病会给免疫功能低下的患者带来相当大的发病率和死亡率，但该疾病的流行病学尚未得到充分研究，也没有针对任何病原体描述标准的分子分型方法。在这里，我们描述了一种米根霉的多位点微卫星分型（MLMT）方法。从真菌基因组倡议数据库（Broad Institute）下载米根霉基因组序列。借助在线重复搜索工具 Repeat Masker，筛选了大约 570 万个碱基对的基因间区和 ORFs 的重复区。在验证了 30 个微卫星位点中的 3 个（RO3，（CCT）（n）；RO4，（TA）（n）；和 RO8，（GAA）（GGA）（n））能够扩增和在 8 个随机选择的米根霉临床分离株中检测到它们的大小变化后，选择了这 3 个微卫星。这些位点的核苷酸序列分析显示微卫星重复数存在多态性。基于使用荧光标记引物在自动毛细管电泳中根据片段大小确定，在 30 个临床分离株上评估了这些微卫星位点的菌株分化能力。发现这 3 个多态性微卫星位点具有良好的区分能力（D）（RO3，D=0.846；RO4，D=0.747；RO8，D=0.742；合并 D=0.986）和 7 次亚培养的稳定性。还证实 MLMT 方法可用于米根霉和德氏根霉（一种拟议的新种），尽管 MLMT 分析不能将它们分为两个聚类。这里描述的 MLMT 系统，据信是首次用于接合菌，有望成为米根霉菌株分型的有力工具。
