Ahmed A O, Mukhtar M M, Kools-Sijmons M, Fahal A H, de Hoog S, van den Ende B G, Zijlstra E E, Verbrugh H, Abugroun E S, Elhassan A M, van Belkum A
Institute of Endemic Diseases, University of Khartoum, Sudan, The Netherlands.
J Clin Microbiol. 1999 Oct;37(10):3175-8. doi: 10.1128/JCM.37.10.3175-3178.1999.
Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.
马杜拉足分支菌是苏丹和热带非洲其他国家真性足菌肿最常见的病因。目前，足菌肿的早期诊断较为困难。为了提高马杜拉足分支菌的鉴定能力，进而改善足菌肿的诊断，我们基于编码真菌核糖体RNA的基因之间的内转录间隔区（ITS）序列开发了特异性寡核苷酸引物。用通用引物扩增ITS区域并进行测序，然后设计了两组物种特异性引物，它们能特异性扩增ITS和5.8S核糖体DNA基因的部分片段。用从人类足菌肿病变中分离的DNA、从马杜拉足分支菌参考菌株及相关真菌培养物中提取的DNA以及人类DNA对新引物的特异性进行了检测。为研究马杜拉足分支菌ITS区域的遗传变异性，从25个不同的临床分离株中获得ITS扩增子，并用CfoI、HaeIII、MspI、Sau3AI、RsaI和SpeI限制性内切酶进行限制性片段长度多态性（RFLP）分析。ITS区域的RFLP分析未显示出任何差异，表明在本研究中分析的分离株具有同质性。
