Development of a Species-Specific PCR-Restriction Fragment Length Polymorphism Analysis Procedure for Identification of Madurella mycetomatis

Abstract

ABSTRACT 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 Cfo I, Hae III, Msp I, Sau 3AI, Rsa I, and Spe I 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.