Polymerase Chain Reaction: Restriction Fragment Length Polymorphism Differentiates the Environmental and Clinically Important Fungal Isolates

Abstract

Soil rich in keratinous material represents the main reservoir of keratinophilic fungi. Majority of soil saprophytes are known to cause fungal disease (mycoses) in human beings and animals. Considering the importance of soil saprophytes as potential human pathogens, this study aimed at assessing their presence in various soil samples collected from different locations of Jammu, India. Soil samples were screened for the isolation of keratinophilic fungi by using hair bait technique. A total of 16 fungal isolates were identified on the basis of macro and microscopic characteristics. Out of these, 12 fungal isolates were obtained from soil and four were clinical isolates. PCR–RFLP approach was used for detecting genetic relatedness between the environmental and clinical isolates. ITS (ITS1-5.8S-ITS2) region of 16 fungal isolate was amplified with primer pair ITS1 and ITS4. Gel electrophoresis after PCR of ITS region showed that DNA from all the fungal isolates yielded single fragment ranging from 550 to 650 bp. This was followed by Seminested PCR to detect variations in ITS2 region using primer pair ITS86 and ITS4. The PCR product was subjected to restriction digestion with ten restriction enzymes out of which four enzymes MboI, MseI, MvaI, HpaII showed best results. Restriction fragments obtained were scored for the presence and absence of bands. Dendrogram was generated which divided the fungal isolates into four main groups. PCR-RFLP made possible the detection of interspecies and intrastrainal variations in keratinophilic fungi isolated from soil and clinically relevant isolates from patients with opportunistic infections.