Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identification of Madurella mycetomatis

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and oth er countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomati s and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed s pacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs, T he ITS regions were amplified with universal primers and sequenced, and the n two sets of species-specific primers were designed which specifically amp lify 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 D NA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA, To study the genetic variability of the ITS reg ions of M. mycetomatis, ITS amplicons were obtained from 25 different clini cal isolates and subjected to restriction fragment length polymorphism (RFL P) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enz ymes. RFLP analysis of the ITS region did not reveal even a single differen ce, indicating the homogeneity of the isolates analyzed during the current study.