DEVELOPMENT OF A NEW PNA-FISH BASED METHOD FOR THE SPECIFIC IDENTIFICATION OF
Aspergillus fumigatus.
Author* LAURA CERQUEIRA
Supervisors: Maria João Vieira, Nuno Azevedo
University of Minho
School of Engineering
CEB – Centre of Biological Engineering
* [email protected]
Introduction
Table 1 – Results of A. fumigatus probe specificity test
FISH protocol:
Aspergillus fumigatus is a saprophyte filamentous fungus that feeds
Strains tested
on decaying organic matter (Dagenais and Keller 2009) and produces
Epifluorescence microscopy
(or flow cytometry)
Sample
conidia, which can survive in a wide range of aggressive
environments (Abad et al. 2010). Depending on the host immunologic
system (Dagenais and Keller 2009), the inhaled conidia can be
Detection
FIXATION
Application of chemical fixatives
(formalin, paraformaldehyde and
ethanol)
determinative of disease (Invasive Aspergillosis) especially in
WASHING
Permeabilized cells
All loosely bound or unbound labelled
probes are removed from the sample
providing specificity to the detection
immunocompromised patients (McCormick et al. 2010).
After deposition in the pulmonary space, A. fumigatus may start a
pathogenic behavior in vulnerable hosts by epithelial tissue adherence
and endocytosis. Within epithelial cells, conidia start swelling and
HYBRIDIZATION
Temperature, pH, ionic strength and
formamide concentrations
The probe accesses and hybridizes with the target sequence on
the rRNA of the cell
begin to germinate. The germinated hyphae can escape from the
epithelial cells and infiltrate blood vessels and induce endothelial cell
FIG. 1 – Squematic PNA-FISH proceedment.
damage (McCormick et al. 2010).
Results and Discussion
In here we describe the development of a new fluorescent-labelled
As expected, for the optimized hybridization conditions, the probe only
PNA probe for the specific detection of Aspergillus fumigatus by
hybridized with Aspergillus fumigatus strains (Table 1).
Fluorescence in situ hybridization (FISH). PNA probes are synthetic
Therefore, in practical terms, specificity and sensitivity) were 100%
DNA mimics that have a modified negatively charged chemical
showing the good quality of the selected sequence regarding the capacity
structure although specific hybridization between the PNA and nucleic
of discriminating A. fumigatus among other strains.
acid complementary sequences still occurs according to the WatsonCrick rules. PNA probes normally have smaller sequences (13-18
nucleotides) than DNA sequences (at least 18 nucleotides), higher
thermal stability and a greater resistance to nucleases and proteases
than DNA molecules (Stender et al. 2002). Several PNA probes have
been developed and optimized for a wide range of microorganisms,
FIG. 2 – Epifluorescence microscope visualization of A. fumigatus ATCC 46645.
Visualization of the same microscopic field at the green channel (negative control
of FUM628) (B). Images were obtained with equal exposure times.
including bacteria, Candida species and filamentous fungi.
Methods
•Specificity
and
sensitivity
determination
was
accessed
using
Aspergillus fumigatus strains and other microorganisms that can be
related with pulmonary diseases, by fluorescence microscopy.
•The hybridization procedure is represented in Figure 1.
-Abad A, Victoria Fernandez-Molina J, Bikandi J, Ramirez A, Margareto J, Sendino J, Luis Hernando F, Ponton J,
Garaizar J, Rementeria A. 2010. “What makes Aspergillus fumigatus a successful pathogen? Genes and
molecules involved in invasive aspergillosis.” Rev Iberoam Micol, 27(4):155-182.
-Dagenais TR and Keller NP. 2009. „Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis”. Clin
Microbiol Rev, 22(3):447-465.
-McCormick A, Loeffler J, Ebel F. 2010. ”Aspergillus fumigatus: contours of an opportunistic human pathogen.”
Cell Microbiol, 12(11):1535-1543.
-Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J: PNA for rapid microbiology. J Microbiol Methods 2002,
48(1):1-17.
PNA-FISH
Aspergillus fumigatus MUM 02.24………….………….+
Aspergillus fumigatus MUM 07.05……….…………….+
Aspergillus fumigatus MUM 9802..…....……………….+
Aspergillus fumigatus ATCC 46645 ………...…………+
Aspergillus fumigatus CECT 2071.…….….………...….+
Aspergillus fumigatus CECT 20190..…….......……..…..+
Aspergillus fumigatus CECT 20228..……...…………....+
Aspergillus fumigatus CECT 20366.……………...….....+
Aspergillus ibericus MUM 03.49……….……...…..……Aspergillus ochraceus MUM 9302……..……….……….Aspergillus clavatus MUM 9717………….………...…...Aspergillus versicolor MUM 00.20……………..…..…...Aspergillus terreus MUM 9409.. ………………….….....Aspergillus tubingensisMUM 06.152.…..…………..…...Aspergillus oryzae MUM 10242 ………..…………..…...Aspergillus flavus MUM 00.06 …………..…………..….Aspergillus flavus MUM 9201.. …………..…………..…Aspergillus niger MUM 92.13.……..……………...…….Aspergillus niger MUM 01.01.…….………………..…....Emericella nidulans var. echinulata MUM 9832….......…Neosartorya fisheri var. glabra MUM 9836 ……...…..…Penicillium brevicompactum MUM 02.12 …….…...……Penicillium chrysogenum MUM 061.70 …………........…Mucor hiemalis MUM 9732. ……………….………….....Trichoderma viride MUM 9754.……….…………......…..Candida parapsilosis ATCC 22019..……………………..Candida tropicalis ATCC 750...…………………...……..Candida glabrata ATCC 2001..…………...…….………..Candida albicans ATCC 1472..…………………………..Pseudomonas aeruginosa PAO1……….…………………Pseudomonas aeruginosa CECT 111..……….…………...Escherichia coli K12 …………………………….……….Staphylococcus aureus CECT 239..……………………….-
Conclusion
In here, a new molecular diagnostic method is proposed using a specific
peptide nucleic acid (PNA) probe for direct visualization of A. fumigatus
by fluorescence in situ hybridization (FISH), in a very specific and
sensitive way.
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