Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.
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 (10¹ 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 10² spores and 75 % for 10¹ 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.
Nawrot
Urszula
U
Department of Microbiology, Wroclaw Medical University, Poland. nawrot@mbio.am.wroc.pl
Wlodarczyk
Katarzyna
K
Wrobel
Magdalena
M
Wasik
Anita
A
Dobosz
Tadeusz
T
D001232:Aspergillus fumigatus / Q000235:genetics*; D001769:Blood / Q000382:microbiology; D004271:DNA, Fungal / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D013172:Spores, Fungal / Q000235:genetics*; D014871:Water Microbiology
D004271:DNA, Fungal; D011933:Reagent Kits, Diagnostic