Automated and manual methods of DNA extraction for Aspergillus fumigatus and Rhizopus oryzae analyzed by quantitative real-time PCR

Abstract

Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules and germinated hyphal elements (GHE). The limit of detection of A. fumigatus and R. oryzae GHE in bronchoalveolar lavage (BAL) fluid with either extraction method was 1 GHE/ml. Both methods efficiently extracted DNA from A. fumigatus, with a limit of detection of 1 x 10(2) conidia. Extraction of R. oryzae by the manual method resulted in a limit of detection of 1 x 10(3) sporangiospores. However, extraction with the automated method resulted in a limit of detection of 1 x 10(1) sporangiospores. The amount of time to process 24 samples by the automated method was 2.5 h prior to transferring for automation, 1.3 h of automation, and 10 min postautomation, resulting in a total time of 4 h. The total time required for the manual method was 5.25 h. The automated and manual methods were similar in sensitivity for DNA extraction from A. fumigatus conidia and GHE. For R. oryzae, the automated method was more sensitive for DNA extraction of sporangiospores, while the manual method was more sensitive for GHE in BAL fluid.

FIG. 1.

Amounts of DNA extracted by the automated and manual methods of DNA extraction. (A) Extraction of 10-fold serial dilutions of Aspergillus fumigatus. a, not significant; b, P = 0.0003. (B) Extraction of Aspergillus fumigatus GHE in normal rabbit BAL fluid. a, not significant; b, P ≤ 0.008. (C) Extraction of 10-fold serial dilutions of Rhizopus oryzae sporangiospores. a, b, and c, P ≤ 0.008. (D) Extraction of Rhizopus oryzae GHE in normal rabbit BAL fluid. The manual method extracted significantly more DNA (a, P = 0.0006) at a level of detection of 1 × 10⁰ GHE than the automated method at the same level.