Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.

Abstract

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10 1 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10 2 spores and 75 % for 10 1 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/ Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.

DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies

“…On the one hand, the fact that the Qiagen kit was effective for the extraction of A . fumigatus spores was not surprising, as this has been already shown in studies focused on the diagnosis of invasive aspergillosis 49 . We confirmed this result through quantification, but also with a sequencing-based experiment, showing that a higher relative abundance of A .…”

“…Its effectiveness for the extraction of fungal DNA has been shown in one study 25 , and our initial hypothesis was that by adding an additional bead beating step, we could improve the extraction of fungal DNA. The effectiveness of bead beating on sturdy fungal structures including spores was observed in other studies (in combination with other methods/kits) 48,49 and it is considered a better alternative than the use of an enzymatic step, because of the lower cost and shorter processing time 50 . It was interesting to see that this was observed only for two species ( C .…”

Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

“…Third, the dissociation capacity of lysis buffer is generally insufficient to completely disrupt the cell wall of filamentous fungi or cystic stages of parasites. In these cases, the mechanical disruption of cells notably improves the extraction yield and gives better results than the enzymatic treatment with PK, thereby considerably enhancing the detection sensitivity of such pathogens as mentioned by Nawrot [ 18 ]. Although vortexing with glass beads was sufficient for yeast treatment, filamentous fungi extraction yield was only enhanced using high-power mechanical grinding.…”

Comparison of four automated nucleic acid extraction platforms for the recovery of DNA from Aspergillus fumigatus

“…The majority of these papers document in-house assays using differing extraction and amplification techniques, highlighting the reasons for excluding PCR from these disease-defining criteria (De Pauw et al ., 2008). Over the last 10 years, increasing efforts have been made to standardize Aspergillus PCR through the establishment of several consensus groups and an ever-growing availability of PCR and extraction comparisons (Francesconi et al ., 2008; Fredricks et al ., 2005; Griffiths et al ., 2006; Karakousis et al ., 2006; Nawrot et al ., 2010).…”

“…red and white cell lysis and bead beating) and elution volume all affected PCR performance, and led to the conclusion that Aspergillus PCR efficiency is limited by extraction and not by PCR amplification (White et al ., 2010a, b). This confirmed other research in which conventional extraction methods suitable for extracting nucleic acid (NA) from bacteria and viruses were shown to be insufficient for NA isolation from fungi, and bead beating and increased sample input volume (>600 µl) were suggested as essential (Francesconi et al ., 2008; Griffiths et al ., 2006; Nawrot et al ., 2010). A degree of diagnostic molecular standardization can be achieved by using automated extraction platforms with commercial quality-controlled kits, minimizing inter-operator variability and hands-on time.…”