Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1/D2 region sufficient for identification of clinical fungi?

Ref ID: 19428

Author:

N. Kiraz,1 Y. Oz,2 H. Aslan,3 Z. Erturan,4 B. Ener,5
S. Arikan-Akdagli,6 M. H. Muslumanoglu7 and Z. Cetinkaya8

Author address:

1Istanbul University Cerrahpasa Medical Faculty, Department of
Microbiology, Istanbul, Turkey; 3Adana Numune Research and
Education Hospital, Department of Medical Genetics, Adana,
Turkey; 4Istanbul University Medical Faculty, Department of
Micro

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives Conventional identification of pathogenic fungi based
on the combination of tests evaluating their morphologic and
chemical characteristics in clinical laboratories, they are time con-
suming and labor-intensive. Also commercially available yeast
identification systems are often insufficient in the identification of
infrequent species or the differentiation of closely related species. A
new molecular diagnostic approach is PCR amplification of target
DNA and then sequencing of amplicons. For this purpose, the
sequence analysis of D1/D2 or ITS regions in ribosomal RNA large
subunit (rLSU) gene has been used in identification of various
yeasts and molds. In our study, we evaluated the DNA extraction
by Whatman FTA filter matrix technology and then identification
by DNA sequencing of D1/D2 region in rLSU of clinical yeasts and
molds.
Methods A total of 1323 clinical yeast and 160 mold isolates were
included in this study. At the beginning, yeast identification was
made by conventional (macroscopic and microscopic morphology,
germ tube production) and commercial (API 20C, Biomerieux and
CHROMagar Candida, BD Diagnostics) tests, molds were identified by
macroscopic and microscopic characteristics. Fungal DNA extraction
was made by using Whatman FTA microcards with indicator. The
amplification of genomic DNA was carried out by nested PCR for
yeasts and seminested PCR for molds. Amplification success was eval-
uated by%1.2 agarose jel electrophoresis. BigDye terminator cycle
sequencing Ready Reaction kits (Applied Biosystems) were used for
cycle sequencing reactions. Resulting products were then analyzed
on an automated capillary DNA sequencer (ABI Prism 3130 Genetic
Analyzer, Applied Biosystems). Isolates were formally identified by
using BLAST (Basic Local Alignment Search Tool) searches against
fungal sequences in existing DNA databases.
Results Unidentified 20 yeasts (Candida and Geotrichum species) and
all of the molds isolates (124 Aspergillus, 19 Fusarium, 6 Trichophy-
ton, 3 Scedosporium, 2 Alternaria, 1 Rhizopus, 1 Mucor, 1 Paecilomy-
ces, 1 Hortaea, 1 Acremonium, 1 Microsporum identified by
microscopically and macroscopically) were included to sequence
analysis. Also 6 quality control isolates were used. Whatman FTA
filter matrix technology provided an easy and extremely rapid
method of preparing both yeast and mould genomic DNAs. Ampli-
con lengths from sequencing were 392-578 bp for yeasts, 404-
628 bp for molds. Conventional identification results were compati-
ble with molecular identification results in general. Yeasts were
identified with 100% identity. The sequences of 35 isolates in Asper-
gillus genus were shown 100% identity both A. flavus and A. ory-
zae. Ten Fusarium isolates exhibited 100% identity with
F.subglutinans and F.proliferatum. Though Trichophyton species could
not be identified by sequencing, Microsporum isolates were shown
100% identity.
Conclusion Whatman FTA matrix technology is an extremely rapid,
practical and successful method for fungal DNA extraction. Although
conventional and commercial phenotypic methods are substantially
sufficient for identification of clinical important fungi, DNA sequence
analysis may be a reliable alternative for some special or uncommon
fungi. Sequence analysis of rLSU rRNA gene D1/D2 region was
found considerably successful in identification of many clinical fungi.
However addition of ITS regions may require for identification of closely related fungi.

Abstract Number: p231

Conference Year: 2013

Link to conference website: NULL

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