A Systematic Comparison of Pre-Treatment Procedures and DNA Extraction for the Standardization of Aspergillosis Real-Time PCR Diagnostic

Ref ID: 19475

Author:

B Greiderer-Kleinlercher1*, P Staudinger1, HW Ott2, E Fréalle3, S Perkhofer4

Author address:

1Department of Biomedical Science, University of Applied Sciences Tyrol, Innsbruck, Austria
2Department of Hemostasis, University Hospital Munich, Munich, Germany
3Parasitology-Mycology Laboratory, University Hospital Center & Pasteur Institute of

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
Standardization of molecular diagnosis of invasive aspergillosis is still a problem in clinical routine
practice. Critical steps in real-time PCR analysis are fungal cell wall disruption and DNA extraction.
The aim of this study was to investigate enzymatic, chemical and physical cell wall disruption
procedures onto efficiency and applicability as a routine diagnostic technique and since, numerous
DNA extraction kits have been tested before, yet under different conditions and pre-treatment
methods. We searched on a panel of twelve different extraction methods which are commonly used
in routine diagnostic and tested them under the same conditions.
Then we used promising DNA extraction kits revealing best results as well as the automated method
with spiked whole blood samples from healthy volunteers to mimic the in vivo situation.
Methods:
A total number of ten different pre-treatment methods for fungal cell wall disruption and twelve
DNA extraction kits including automated DNA extraction with the MagNA Pure LC instrument
were tested for efficiency and specificity for conidia suspensions at three different concentrations
(103, 105 and 107 conidia/ml of Aspergillus fumigatus).
One pre-treatment method and the five promising DNA extraction kits revealing best results plus
automated method were further used with spiked (103, 105, 107 conidia/ml) whole blood samples.
Real-time PCR targeting A. fumigatus 18S rRNA gene was used. Samples without A. fumigatus
served as negative controls.
Results:
Bead beating with Precellys® glass beads (diameter 0.5 mm) was identified as the best cell wall
disruption method giving highest Aspergillus DNA amounts.
Five DNA purification kits revealed statistically higher DNA amount at 103 conidia /ml than the
other kits. Two out of these five kits showed also statistically significant higher efficiency for the
concentration of 105 and three extraction kits showed statistically significant higher efficiency at
107 conidia/ml.
For spiked whole blood samples, the automated DNA extraction method showed statistically
significant higher amounts of DNA than the five other tested kits in all three concentrations and
statistically significant lowest results in negative controls.
Conclusion:
The pre-treatment method revealing best results was bead beating with Precellys® glass beads.
The comparison of the different bead materials identified glass as material with the lowest binding
capacity for nucleic acids as it is approved from data on RNA extraction from filamentous fungi.
– 72 –
A parameter which is important to gain useful results with PCR, is the ability to efficiently isolate
fungal DNA. Several studies on fungal DNA extraction kits have been performed and published yet
under different conditions and different pre-treatment methods which make comparison difficult.
In this study the automated DNA extraction method showed best results for spiked blood samples
negative controls. The clear advantage of the MagNA Pure LC instrument is the fully automated
system. It is possible to time efficiently isolate many samples in a standardized manner with low
contamination risk.
It is suggested that the fungal burden in the bloodstream of patients is low. Therefore, a high degree
of analytical sensitivity is needed to detect these small amounts of DNA to avoid false negative
samples.

Abstract Number: 3

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

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