Evaluation of three DNA extraction methods from fungal cultures

Abstract

Polymerase chain reaction (PCR) based assays have been developed to amplify DNA of fungal pathogens as culture-based detection methods show low sensitivity. In order to perform a sensitive, specific, and reliable PCR based assay, the availability of pure DNA as well as an easy-to-perform DNA extraction protocol is essential. The existing protocols for DNA extraction used for bacteria or viruses show poor release of fungal DNA. In this study, we evaluated three different methods of DNA extraction and compared their efficacy in the extraction of DNA from filamentous fungi, yeasts, and dermatophytes commonly isolated in our laboratory. It was found that the Fungi/Yeast Genomic DNA Isolation Kit (Norgen Biotek Corp, Ontario, Canada) demonstrated satisfactory extraction of DNA from all the fungi analyzed as compared to that of the Qiamp DNA extraction kit (Qiagen GmbH, Dusseldorf, Germany) or the Phenol Chloroform Isoamyl alcohol extraction method which failed to extract amplifiable DNA from many of the fungal species. Thus, we recommend the use of Fungi/Yeast Genomic DNA Isolation Kit (Norgen) with modifications for the extraction of DNA from fungal cultures.

Keywords: DNA extraction methods; Fungi; Polymerase chain reaction.

Fig. 1

Gel electrophoresis of amplicons generated from DNA extracted by: (Panel 1a and 1b) Fungi/Yeast Genomic DNA Isolation Kit (Norgen). (Panel 2) Qiagen DNA mini kit (QIAamp, Qiagen). (Panel 3) Phenol chloroform iso-amyl alcohol method. C.alb – Candida albicans, C.trop – Candida tropicalis, C.neo – Cryptococcus neoformans; T.asa – Trichosporon asahii, Pen – Penicillium spp., Fus – Fusarium spp., Epid – Epidermophyton mentagrophytes, M.gyp – Microsporum gypseum, A.fum – Aspergillus fumigatus, Rhizopus – Rhizopus spp., Fusariium – Fusarium spp., M.gyp – Microsporum gypseum, E.mento – Epidermophyton mentagrophytes, Penicillium – Penicillium spp., NC – negative control, MM – 100 bp molecular marker (500 bp is marked).