Molecular detection of Aspergillus fumigatus and section Fumigati by multiplex Real Time PCR using ITS and 946;-tubuline sequences

Ref ID: 19533

Author:

JV Fernandez-Molina1*, A Abad-Diaz-de-Cerio1, M Sueiro-Olivares1, A Pellón1, A Ramirez-
Garcia2, J Garaizar2, J Pemán3, FL Hernando1, A Rementeria1

Author address:

1Immunology, Microbiology and Parasitology, Faculty of Sciences and Technology, UPV/EHU, Leioa, Spain
2Immunology, Microbiology and Parasitology, Faculty of Pharmacy, UPV/EHU, Vitoria, Spain
3Microbiology, Hospital of La Fé, Valencia, Spain

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
The aim of this work was to develop a multiplex real time PCR to quickly and easily distinguish
species of section Fumigati from other fungi and specifically identify A. fumigatus in one step,
avoiding PCR inhibitions.
Methods:
We designed primers and specific Taqman probes using as a target internal transcribed spacer regions
and β -tubulin gene, to detect members of section Fumigati and A. fumigatus species, respectively.
We also designed an internal control, with specific primers and a Taqman probe, to detect inhibition
of the reactions. Finally, several PCR facilitators was studied and included in PCR mixtures to avoid
false negatives due to PCR inhibitions. The sensitivity and efficiency of the multiplex real time
PCR assay was tested with DNA extracted from hyphae and conidia from A. fumigatus AF-293 and
from a collection of 67 different strains. Moreover, we checked the performance of the method with
38 human bronchoalveolar lavage samples.
Results:
The multiplex real time PCR was optimized using genomic DNA extracted from A. fumigatus AF-
293 showing sensitivity and mean efficiency of 20 fg and 82.16%, respectively, in the detection of
section Fumigati; and 50 fg and 101.23% in the specific detection of A. fumigatus. Furthermore,
the method developed also allows the quantification from 20 ng to 200 fg of A. fumigatus genomic
DNA in samples and, in particular, detection of germinated conidia (down to 1 germinated conidium
vs. 103 non-germinated conidia). In addition, the method was successfully tested on a collection
of microorganisms and on several bronchoalveolar lavage samples. On the other hand, we also
included an internal control and another specific Taqman probe for its detection to analyze the PCR
inhibition. The inclusion of some PCR facilitators (polyethylene glycol and BSA) together with
the dilution of samples (1:2) made possible to avoid false negatives due to PCR inhibitions in all
bronchoalveolar lavages assayed.
Conclusion:
The multiplex real time PCR assay developed in this study has demonstrated to be an excellent
method to detect DNA of A. fumigatus in bronchoalveolar lavage samples, even though the
Fumigati probes designed do not provide the best results. In addition, we confirmed the detection
of hyphae and germinated conidia (even a single one), but not non-germinated conidia (below of
103 conidia/ml). Finally, we were able to avoid the PCR inhibition when the reaction was applied to
human samples.
NOTE: THIS ABSTRACT HAS BEEN SELECTED FOR ORAL PRESENTATION.

Abstract Number: 60

Conference Year: 2014

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

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