Detection and Differentiation of Causative Fungi of Onychomycosis with PCR-RFLP

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Objective Using polymerase chain reaction(PCR)restriction fragment length polymorphism (RFLP) to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis,and to decrease the time taken for the laboratory identification of pathogens.Methods Fungi species and nails were analyzed by polymerase chain reaction(PCR),primer systems were designed in conserved sequences of the small ribosomal subunit 18S rRNA genes shared by most fungi,and differentiated between species by HaeⅢ digestion of the amplified product.The results were compared with that of conventional KOH preparation and fungal culture.Results Fragments of the gene coding for 18S rRNA were amplified successfully from all fungi species,HaeⅢ digestion of the PCR products were sufficiently different to allow the differentiation of dermatophyte,yeast,and mold.Positive PCR results were obtained from all 15 dystrophic nails,with the positive rates of other methods were 10/15 of KOH preparation,and 9/15 of fungal culture.Normal nails were all PCR amplification negative.In 9 positive samples confirmed by culture,we just got the same result through the PCR RFLP.Conclusion PCR analysis of nail specimens infected with fungus appears to be a more sensitive diagnositic method for onychomycosis than that of KOH preparation and culture techniques.HaeⅢ digestion of the PCR products seems a useful step in differentiating dermatophytes,yeasts,and molds.

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