ELUCIDATING THE DERMATOPHYTE SPECTRUM THROUGH RAPID MULTIPLEX PCR AND DETECTION OF ANTIFUNGAL DRUG RESISTANCE CAUSED BY MUTATIONS IN SQUALENE EPOXIDASE AND 14-ALFA DEMETHYLASE GENE

All clinically identified cases of dermatophyte skin infections not on treatment with topical and/or systemic antifungal from 2 weeks prior will be included in the study. Skin scrapings will be collected using no.15 sterile blade on the Dermatophyte Test Media and KOH mount will be done.  The media that shows growth of filamentous fungi will be then subcultured on to SDA for species identification.

A portion of the skin scrapings (~100 mg) will be aliquoted for DNA extraction. Fungal DNA will be extracted from the skin scraping . The extracted DNA will be quantified using a

Nanodrop UV spectrophotometer. Conventional PCR and Species specific RT PCR will be performed . It is performed for live fungi where protein synthesis is taking place. PCR products will be resolved by horizontal gel electrophoresis and results will be analyzed using the gel documentation system. The amplicons will be purified and then sequenced. Sequences will be analyzed using bioinformatics tools to identify

species and determine the clonal nature of the fungi detected. Squalene epoxidase gene will be amplified using three sets of primers. The sequences will then be analyzed for point mutations using bioinformatics tools. Primers for CYP51 gene which codes for 14α-demethylase will be designed. Cycling conditions will be optimized.This target gene will be amplified and sequenced.

The sequences will then be analyzed for mutations using bioinformatics tools.