A simple and inexpensive method for genomic DNA extraction from fungi using Whatman FTA9415; Elute filter paper for conventional and real-time PCR assays

R.L. Gorton1, T.A. Vyzantiadis2, S. Seaton1, C.C. Kibbler1

Author address: 

1The Royal Free NHS Trust, Hampstead, LONDON, United Kingdom 2Aristotle University, THESSALONIKI, Greece

Abstract: 

Objectives: DNA extraction using filter paper has become well established and is suitable for conventional but not real time PCR protocols. Whatman FTA® Elute paper offers a simplified method for the extraction of DNA from fungal cultures. The extracted DNA is eluted from the FTA® matrix into sterile water. Our objective was to evaluate the FTA® Elute paper’s ability to extract DNA from filamentous fungi and assess the performance of the DNA eluate with conventional and real-time PCR. Method: A total of 75 moulds and 10 yeasts were collected from clinical samples, of which 70 moulds were sub-cultured in glucose/yeast/peptone (GYP) broth and 5 moulds and 10 yeasts were taken directly from Sabouraud agar. A pea-sized quantity of mycelium/yeast was freeze/thawed and ribolysed in molecular grade water and then applied to the FTA® Elute card. To inactivate the fungal mass, the card was dried at 80°C for 20 minutes. Elution of the genomic DNA from a 3mm punch of FTA® Elute card into 50μl of sterile water was achieved by heating at 95°C for 30 minutes. Elutions were performed at day 0, weeks 4 and 12 to evaluate the preservation of DNA on the paper. A pan-fungal ITS (internal transcribed spacer) rDNA PCR assay was used to amplify the eluted DNA and the amplicons were sequenced to confirm the phenotypic identification of each fungus. By converting the conventional ITS rDNA PCR the amplification of DNA from the eluate was evaluated with real-time PCR on the Corbett Rotor-Gene 6000, and high resolution melt (HRM) curve analysis was performed on the ITS rDNA amplicons. Results: The Whatman FTA® Elute paper successfully extracted DNA from all the fungal cultures from GYP broth and Sabourauds agar including Candida spp, Aspergillus spp, Fusarium spp, Scedosporium spp, Acremonium spp and Zygomycetes (n=60). Using a 3mm punch of Whatman FTA® Elute paper the quantity of DNA eluted ranged between 50ng to 200ng of DNA per punch. The ITS rDNA region was amplified from all the DNA extracts using the conventional PCR (n=75) and all isolates were identified genotypically using the NCBI Blast Nucleotide database. Real-time PCR amplification of the ITS rDNA gene from the DNA elutions was successful. Furthermore the HRM analysis revealed a significant difference in the melting temperatures of the ITS rDNA amplicons for the different fungal species (complete real-time data will be presented in the poster). Re-elution of the DNA at weeks 4 and 12 were successful and quantification using a NanoDrop® ND-1000 showed no evidence of degradation of DNA over time. Conclusion: Whatman FTA® Elute paper provides a simple, inexpensive and chemical free method of genomic DNA extraction from yeasts and moulds. The ability to elute DNA enables this extraction method to be used with real-time PCR assays in place of laborious and timeconsuming post-PCR analysis for amplicon detection. The ability to archive the papers provides the user with an invaluable source of genomic DNA for future use.
2009

abstract No: 

P222

Full conference title: 

4th Trends in Medical Mycology
    • TIMM 4th (2012)