Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR

Abstract

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze-thaw method, a freeze-boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR

“…Therefore, in addition to some type of enzymatic and or mechanical pretreatment, modifications of kit protocols may be necessary (11,18,26). The manual DNA extraction methods described in the literature for filamentous fungi tend to be labor-intensive and time-consuming, rendering them unsuitable for high-throughput diagnostic laboratories (8,9,11,13,18,21,22,25,26). When small numbers of samples need to be processed, our laboratory uses the manual DNA extraction method incorporating enzymatic pretreatment and mechanical disruption with a modified version of the DNeasy Plant minikit protocol (25,26).…”

“…The automated or manual method may be suitable for DNA The evolution of molecular diagnostics for mycology is advancing with the development of fully automated platforms which have the ability to extract DNA from fungi (8,13,19,21). We investigated the MagNA Pure LC instrument, a widely used fully automated closed system, for DNA extraction.…”

“…These methods are time-consuming and therefore reduce the ability for rapid diagnosis. The efficiency of extraction of fungal DNA may vary considerably depending on the method chosen (9,11,13,15,25). Thus, the extraction method chosen may often represent a compromise between efficiency, lack of exogenous contamination, and the ability to be adapted by routine high-throughput laboratories (15).…”

Single-Dose Pharmacodynamics of Amphotericin B against Aspergillus Species in an In Vitro Pharmacokinetic/Pharmacodynamic Model

“…Aspergillus DNA was extracted from 200-l samples from the IC of the in vitro PK/PD model after 0 and 72 h with the Qiagen DNA blood minikit (Roche Diagnostics, Athens, Greece) after enzymatic (incubation with proteinase K at 56°C for 10 min) and mechanical (1-min vortexing with glass beads) extraction as previously described (18). Real-time PCR was performed with a previously described assay (2Asp assay) using Aspergillus specific primers (ASF1 and ADR1) and probe (ASP28P) (19).…”

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under MixedEimeriaInfection

“…The development of molecular techniques has streamlined fungal detection and quantification. Communal DNA or molecular probes can be used to target species as well as to facilitate the monitoring of entire fungal communities (Gharizadeh et al, 2004;Goebes et al, 2007;Griffiths et al, 2006;May et al, 2001;Scupham et al, 2006;Urubschurov et al, 2008). Scupham et al (2006) estimated that fungi comprise 0-10%, with a median of 2%, of biota associated with murine cecal biofilm, and that fungi sparsely populated digesta outside the ceca.…”