Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system

Abstract

A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p <or= 0.004). DNA from all fungal species analyzed could be extracted and amplified by real-time PCR. Negative controls from all MagNA Pure isolations remained negative. Sixty-three clinical samples showed identical results by both methods, whereas in 4 of 67 samples, discordant results were obtained. Thus, the MagNA Pure LC technique offers a fast protocol for automated DNA isolation from numerous fungi, revealing high sensitivity and purity.

FIG. 1.

Sensitivity and reproducibility of the MagNAPure LC assay. Serially diluted A. fumigatus conidia (10³ to 10¹ CFU), spiked to 1 ml of blood from healthy donors. Additionally, the sample cartridge was loaded with an alternating positive (A. fumigatus, 10³ CFU, 10 samples) and negative (ddH₂O, 10 samples) pattern to demonstrate the reproducibility and risk of cross-contamination.

FIG. 2.

Extraction of DNA by using the MagNAPure LC system from 23 different yeast and filamentous fungi and amplification by LightCycler. Amplicon analysis by 2% agarose gel electrophoresis. Lanes: 1, 100-bp ladder; 2, Aspergillus fumigatus, 10 CFU; 3, negative control (ddH₂O); 4, negative control (ddH₂O); 5, Aspergillus niger; 6, Aspergillus versicolor; 7, Aspergillus terreus; 8, Alternaria alternata; 9, Paecilomyces variotii; 10, Scopulariopsis brevicaulis; 11, Penicillium brevicompactum; 12, Penicillin chrysogenum; 13, Absidia corymbifera; 14, Fusarium solani; 15, 100-bp ladder; 16, Rhizopus oryzae; 17, Acremonium chrysogenum; 18, Hansenula anomala; 19, Rhodotorula piliruaniae; 20, Trichosporon capitatum; 21, Sporidiobolus johnsonii; 22, Candida albicans; 23, Candida krusei; 24, Candida dublinensis; 25, Candida inconspicua; 26, Candida lusitaniae; 27, Candida glabrata; 28, negative control (ddH₂O).