Development of a Rapid and Low-Cost Method for the Extraction of Dermatophyte DNA.

Abstract

Background:Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate.Aims and Objectives:In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes.Materials and Methods:DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis.Results:Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA.Conclusion:Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions.