Direct Spiecies Identification Of Common Pathogenic Dermatophyte Fungi In Clinical Specimens

Objective:To develop a rapid and reliable molecular biology method to identify the common dermatophyte fungi from clinical specimens.Method:The genome DNA was extracted from 65 cultured strains of 7 common dermatophyte fungi species and part of clinical specimen.Internal transcribed spacer(ITS) region was amplified by semi-nested PCR(snPCR) with 3 universal primers (NS5,ITS1 and ITS4)for fungi.The amplifid products were digested with 2 restriction endonuclease (BciT130 I,Dde I)--RFLP. the rest of each clinical specimen was tested under microscope and cultured in Sabouraud's Agar medium.Then the results of RFLP compared with the cultivation results.Results:The length of ITS regions of 7 common dermatophyte fungi species were similar,from 520bp to 740bp.The digestion of 7 common dermatophyte fungi produced different restriction profiles of their own; Additionally,T mentagrophytes and M gypseum have 2 different band patterns respectively,after 2 endonuclease (BciT130 I,Dde I) restriction. Of 27 clinical specimens that were positive by microscopy,18 ones were positive by culturation(66.7%), 14 ones were dermatophytes,and the rest 4 ones were non-dermatophytes;DNA were extracted from 10 clinical specimens (37.0%);5 clinical specimens were positive by first PCR(18.5%),23 clinical specimens were positive by second PCR (85.2%);Restriction profiles of 17 clinical specimens matched respectively to them of the cultured strains,and 14 profiles of the 17 ones were matched to culturation result completely;3 of the 17 clinical specimens were negative by culturation,but positive by sn-PCR and RFLP.Conclusion:It is impossible to identify the common dermatophyte fungionly by the length of ITS region. Restriction patterns of 7 common dermatophyte fungi were different from each other.T mentagrophytes and M gypseum have 2 different band patterns respectively,after 2 endonuclease (BciT130I,DdeI) restriction. snPCR-RFLP analysis of ribosomal-DNA intergenic spacer regiongs is a valuable method of exactness and celerity for species identification of common dermatophyte fungi from clinical specimens...