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Journal of Clinical Microbiology, July 1998, p. 2057-2062, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Utility of Random Amplified Polymorphic DNA PCR and
TaqMan Automated Detection in Molecular Identification of
Aspergillus fumigatus
Mary E.
Brandt,1,*
Arvind A.
Padhye,1
Leonard W.
Mayer,1 and
Brian P.
Holloway2
Division of Bacterial and Mycotic
Diseases1 and
Biotechnology Core
Facility,2 National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
30333
Received 6 November 1997/Returned for modification 9 January
1998/Accepted 8 April 1998
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ABSTRACT |
We developed a method for the identification of Aspergillus
fumigatus fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City,
Calif.). DNA from seven clinically important Aspergillus species was screened by RAPD-PCR to identify section- or
species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp
product was amplified from all A. fumigatus strains
initially examined but not from other species. A partial DNA sequence
of this product was used to design a specific primer pair, which
generated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a "touchdown" (65
55°C) annealing
protocol was used. The TaqMan system, a fluorogenic assay which uses
the 5'3' endonuclease activity of Taq DNA polymerase,
detected this 864-bp product with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample generated an indeterminate
result. With DNA from three morphologically typical A. fumigatus isolates, six white ("albino") A. fumigatus isolates, and five of six Neosartorya
species (non-A. fumigatus members of the section
Fumigati), the 864-bp product was amplified differentially at an annealing temperature of 56°C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of
heterologous Aspergillus, Penicillium, and
Paecilomyces species or from Neosartorya
fennelliae; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR
and TaqMan assay offers promise as a nucleic acid-based system that can
be used for the identification of filamentous fungal isolates and that
requires no postamplification sample manipulations.
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INTRODUCTION |
Filamentous fungal isolates are
typically identified by microscopic demonstration of characteristic
morphologic structures after growth on appropriate media.
Identification may be delayed if the isolate fails to form the
diagnostically appropriate structures. Furthermore, inexperience in
microscopy may lead to misidentification.
These problems may be obviated by using DNA-based methods for
identification and species assignment of isolates. One approach to
probe design uses random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) to generate markers for any specific genome
(8; for a review, see reference
10). The major advantage of this approach is that no
prior DNA sequence information is required. Likewise, several methods
that detect amplified products exist (for a review, see reference
22), but most require extensive post-PCR sample
manipulation and additional incubation time before results are
available.
The automated TaqMan detection system (Perkin-Elmer Corp., Applied
Biosystems, Foster City, Calif.) is a novel fluorogenic assay for the
detection of PCR products. It takes advantage of the endonuclease
activity of Taq polymerase and Förster-type energy
transfer of a fluorescence-labeled probe (4, 14, 19, 26).
The TaqMan probe consists of a 5' reporter dye (6-carboxyfluorescein [FAM]), a 3' quencher dye (6-carboxytetramethylrhodamine [TAMRA]), and a 3' blocking phosphate group. The fluorescence emission of the
reporter dye is suppressed in the intact probe by Förster-type energy transfer (9). During PCR, the probe is cleaved by the 5' nuclease activity of Taq polymerase only when it is
hybridized to a complementary target. When cleavage between the
reporter and quencher occurs, an increase in reporter dye fluorescence occurs, indicating that the probe-specific PCR product has been generated. Repeated cycles of probe annealing and cleavage result in
exponential amplification of the PCR product and of reporter fluorescence. Fluorescence intensity is measured in an LS50B
luminescence detector (Perkin-Elmer Corp., Applied Biosystems).
The goal of this study was to explore the utility of the RAPD-PCR
screening and TaqMan assay approaches in the identification and
taxonomy of filamentous fungi by using Aspergillus
fumigatus, an agent of invasive aspergillosis, as the target
organism. A RAPD-PCR technique was used to screen A. fumigatus DNA for species-specific amplicons. A specific 1,264-bp
band was amplified from A. fumigatus DNA but not from the
DNA of other species. The DNA sequence of this fragment was determined
and was used to design a specific primer pair, which amplified a single
864-bp fragment from morphologically typical A. fumigatus by
a "touchdown" (6555°C) annealing protocol. We then evaluated
the usefulness of these primers in identifying A. fumigatus
and the TaqMan assay for detection of the amplified product.
(These data were presented in part at the 96th General Meeting of the
American Society for Microbiology, New Orleans, La., 19 to 23 May 1996 [5a].)
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MATERIALS AND METHODS |
Strains and growth conditions.
A total of 164 isolates were
tested in this study. Seventy-one clinical A. fumigatus
isolates and 1 isolate reidentified at the Centers for Disease Control
and Prevention (CDC) as a Neosartorya sp. were received from
San Francisco, Calif., as part of CDC's fungal active surveillance
(1992 to 1994). Nineteen other clinical and environmental A. fumigatus strains, including six nonpigmented ("albino")
A. fumigatus strains, were obtained from the CDC reference culture collection. Five clinical isolates representing morphologic variants of A. fumigatus were supplied by L. J. R. Milne, Regional Mycology Reference Laboratory, Western General
Hospital, Edinburgh, United Kingdom. Five A. fumigatus and
seven Neosartorya strains (23) were provided from
the stock collection of S. Peterson, Northern Regional Research Center,
U.S. Department of Agriculture, Peoria, Ill. Fifty-six isolates
consisting of Penicillium notatum (n = 1),
Penicillium spinulosum (n = 1),
Penicillium citrium (n = 1),
Penicillium funiculosum (n = 1),
Penicillium marneffei (n = 3),
Penicillium (undetermined) species (n = 1),
Paecilomyces variotii (n = 1),
Aspergillus flavus (n = 26),
Aspergillus niger (n = 9), Aspergillus
nidulans (n = 3), Aspergillus glaucus
(n = 1), Aspergillus oryzae
(n = 1), Aspergillus terreus
(n = 4), Aspergillus ustus
(n = 1), and Aspergillus versicolor
(n = 2) were also obtained from the CDC culture
collection; 34 of these came from the San Francisco active
surveillance. Isolates were stored at 
80°C. Working stocks were
maintained on Czapek Dox medium at 4°C, and species identifications
were confirmed by conventional methods (3).
DNA isolation.
A spore suspension from a 7-day culture on
Czapek Dox medium was inoculated into 15 ml of YPD
(yeast-peptone-dextrose) medium in a petri dish, and the dish was
incubated at 30 or 37°C for 48 to 72 h. DNA was then prepared
from the hyphal mat by the Puregene plant tissue protocol (Puregene;
Gentra Systems, Inc., Research Triangle Park, N.C.) according to the
manufacturer's instructions, except that the final DNA pellet was
solubilized in 10 mM Tris-1 mM EDTA-0.7 M NaCl, made to 1% in
cetyltrimethylammonium bromide, and incubated at 60°C for 15 min. The
solution was then extracted with CHCl3, and DNA was
reprecipitated with 2 volumes of ethanol. The DNA concentration of each
sample was determined by using a fluorometer (Hoefer, San Francisco,
Calif.) with calf thymus DNA as a standard. A portion of each DNA
sample was also checked for integrity on an agarose gel before use.
DNA sequencing and analysis.
RAPD was performed with decamer
primers (Operon Z kit; Operon Technologies, Alameda, Calif.) as
described previously (5). The DNA bands of interest were
excised from Tris-acetate gels, purified with GeneClean (Bio 101, Inc.,
La Jolla, Calif.), and cloned with the PCR-Script SK+ cloning kit
(Stratagene, La Jolla, Calif.). Plasmid DNA was purified on a QIA-prep
spin column (Qiagen, Chatsworth, Calif.). DNA sequencing was performed
on an ABI model 373 automated DNA sequencer by using the PRISM Ready
Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer Corp., Applied Biosystems) with double-stranded plasmid DNA templates.
DNA sequences were analyzed by using DNAStar software (DNAStar, Inc.,
Madison, Wis.). PCR primer sequences were selected by using the Oligo
program (24) (version 5; National Biosciences, Plymouth,
Minn.). Two primers, primer Z19-276 (5'-TTGATCTGGCCCTGGCTTGGG) and primer Z19-660 (5'-CAACATTGAAATCCAAGAGGC), were
chosen to amplify the 864-bp A. fumigatus-specific fragment.
The universal fungal primers NS7 and NS8 (27) were used to
coamplify a 377-bp fungal 18S rRNA gene fragment in the same reaction
tube.
Touchdown PCR.
DNA amplifications were performed in a model
9600 DNA thermal cycler (Perkin-Elmer Corp., Applied Biosystems).
Reagents were obtained from Boehringer Mannheim (Indianapolis, Ind.);
AmpliTaq was obtained from Perkin-Elmer Corp. PCR was performed in a
50-µl reaction mixture with final concentrations (per reaction) of
1× PCR Core Buffer, 2.5 mM MgCl2, 0.2 mM (each)
deoxynucleoside triphosphates, 10 pmol of each primer, 1 U of AmpliTaq,
and 50 ng of template. Initial amplifications were performed as 35 cycles of 94°C for 1 min (denaturation), 56°C for 1 min
(annealing), and 72°C for 1 min (extension), followed by a 10-min
final extension at 72°C. Further amplifications were performed by
using a touchdown protocol (7, 17) from 65 to 55°C.
Briefly, the annealing temperature was programmed at 65°C for the
first three cycles. Then, the annealing temperature was lowered in
1-increments every three cycles until 61°C and was then lowered in
2-degree increments from 59 to 55°C, at which 10 final cycles were
performed (total of 31 cycles). No-template controls were included with
every experiment.
TaqMan detection assay.
The TaqMan LS50B detection system
(Perkin-Elmer Corp., Applied Biosystems) was used for automated
fluorogenic detection of the products amplified in the touchdown PCR.
TaqMan probes were designed by following the general rules outlined by
the manufacturer (15) and were synthesized in the
Biotechnology Core Facility, CDC. A probe detecting the A. fumigatus-specific 864-bp amplicon (5'-CTCAACAGTGGATTGGACGTAATCA) contained the reporter dye
FAM covalently attached to the 5' end and the quencher TAMRA attached to the thymidine at position 23. A 3' phosphate was added to block amplification of the probe by Taq polymerase. The 377-bp
rRNA gene control amplicon was detected with another TaqMan probe
(5'-CCTTGGCCGAGAGGTCTGGG) containing the reporter dye
hexachloro-6-carboxyfluorescein (HEX) linked to the 5' end and TAMRA
linked to the 3' end via a linker arm thymidine (Amino-Modifier C6 dT;
Glen Research, Sterling, Va.).
The TaqMan assay was carried out in a 50-µl volume reaction as
described above, with the additions of FAM- and HEX-labeled
probes at 5 pmol each per reaction. All samples except the controls
were tested in
duplicate; the controls were tested in triplicate.
Following touchdown
PCR, a 40-µl aliquot from each well was transferred
to a 96-well
flat-bottom white microtiter plate (Perkin-Elmer
Corp.). The emission
intensities of FAM (518 nm), HEX (556 nm),
and TAMRA (582 nm) were read
in a Perkin-Elmer LS-50B luminescence
spectrophotometer equipped with a
microtiter plate reader. An
increase in emission intensity at 518 nm
(FAM) or 556 nm (HEX)
was seen in the presence of specific template
because of the reporter
dye released as the probe was digested by
Taq polymerase.
Data acquisition and analysis were performed by using the Fluorescence
Data Manager 2-Reporter Multicomponent spreadsheet
and software
according to the manufacturer's instructions (Perkin-Elmer
Corp.). For
each reporter, its emission intensity was divided
by the emission
intensity of the quencher to give a ratio defined
as RQ
+
for samples. The baseline ratio of emission intensities with
the intact
TaqMan probe (RQ
) was determined similarly with a series
of three no-template
controls. Controls containing the FAM probe or the
HEX probe alone
were also incorporated to correct for spillover in the
emission
intensity between the two reporters. Finally, for each
reporter
the average RQ
value for the three no-template
controls was subtracted from
the RQ
+ value for each sample
to give a value defined as
RQ for that
reporter. Nonspecific
fluctuations in fluorescence were normalized
in this calculation by
using the quencher as an internal standard.
Final
RQ results were
expressed as normalized values for FAM
and HEX fluorescence,
respectively. Each normalized value represented
the magnitude of signal
generated during PCR with that probe.
An "indeterminate" category was created to define TaqMan results
that consistently registered outside the positive and negative
clusters. Indeterminate FAM standards were defined as FAM results
from
dilutions of two control templates (
A. versicolor and
A. nidulans) that consistently fell between the two clusters
and
that displayed only faint amounts of the 868-bp product on agarose
gels. For each experiment, the means of the positive, negative,
and
indeterminate standards were calculated. Positive and negative
cutoffs
were then calculated by using the maximum-likelihood approach
as the
point halfway between the means of the positive and indeterminate
standards and halfway between the means of the indeterminate and
negative standards, respectively (
2).
To confirm the TaqMan assay results, 25 µl of each touchdown PCR
product was electrophoresed through a 1.4% agarose gel, stained
with
ethidium bromide (0.5 mg/ml), and photographed.
Nucleotide sequence accession number.
The complete 1,264-bp
sequence of the product amplified by the Z19-RAPD primer has been
deposited with GenBank and has been given the accession no. AF022238.
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RESULTS |
RAPD analysis.
DNAs from a set of isolates comprising
medically important Aspergillus species were screened with
20 decamer primers to identify amplicons that appeared to possess
specificity at the species or section level. A. fumigatus
(n = 3 isolates), A. flavus
(n = 4), A. glaucus (n = 1),
A. nidulans (n = 1), A. terreus
(n = 1), A. ustus (n = 1),
and A. niger (n = 5) were screened (Fig. 1A and data not shown). The primer OPZ19
was selected for further study. A band of 1,264 bp in length was
amplified from 66 randomly selected A. fumigatus strains
with the Z19 primer (Fig. 1A and data not shown). DNAs in this band
from isolates CDC MAS92-229 and CDC MAS92-564 were cloned, generating
the plasmids 19B1 and 19B2, respectively, and sequenced. The sequence
information was used to design a specific primer pair, Z19-276 and
Z19-660, which amplified a DNA fragment of 864 bp from A. fumigatus (Fig. 1B).
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FIG. 1.
DNA from Aspergillus spp. amplified in
duplicate either by RAPD-PCR with the OPZ19 primer (A) or by
conventional PCR with the derived primers Z19-276 and Z19-660 (B).
Lanes 1 and 10, A. fumigatus CDC MAS92-229; lanes 2 and 11, A. fumigatus CDC MAS92-564; lanes 3 and 12, A. fumigatus NRRL 163 (Thom 1911 ex type); lanes 4 and 13, A. fumigatus CDC-B5051; lanes 5 and 14, A. flavus
MAS92-30; lanes 6 and 15, A. terreus MAS92-640; lanes 7 and
16, A. niger MAS92-52; lanes 8 and 17, A. fumigatus MAS93-678; lanes 9 and 18, no-template control. BP, base
pairs.
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A search with the BLAST algorithm shows significant sequence similarity
of the full 1,264-bp sequence with two other sequences.
The 1,264-bp
A. fumigatus sequence has 65% identity with YSCH9332
(
Saccharomyces cerevisiae) at bases 33,685 to 34,043 or 60%
identity
at bases 33,685 to 34,440 and has 67% identity with SPAC1F5
(
Schizosaccharomyces pombe) at bases 26,835 to 27,179. These
sequences contain open
reading frames encoding hypothetical products of
3,744 and 3,655
amino acids.
Touchdown PCR.
The Z19-276 and Z19-660 primer pair was
evaluated for sensitivity and specificity of amplification with DNAs
isolated from members of the genus Aspergillus. After 35 cycles at an annealing temperature of 56°C, a variety of nonspecific
bands was amplified from heterologous DNA (Fig.
2A). A touchdown protocol (annealing temperatures, 65 to 55°C) was implemented to improve the specificity of PCR (7), and it nearly eliminated nonspecific amplicons, as determined by visualization of ethidium bromide-stained gels (Fig.
2B). A panel of DNAs from Aspergillus and
Neosartorya strains comprising members of the section
Fumigati were then tested with the original OPZ19 primer and
the Z19-derived primer pair (Fig. 3).
Under lower-stringency conditions (annealing temperature, 56°C), an
864-bp band could be amplified from all tested members of the section
Fumigati except Neosartorya fennelliae (Fig. 3B). However, when identical reagents were used with the touchdown protocol,
an intense 864-bp band was amplified only from strains of A. fumigatus (including A. fumigatus var.
ellipticus, A. fumigatus var. helvola,
and A. fumigatus var. acolumnaris) (Fig. 3C). Two Neosartorya fischeri strains and Neosartorya
glabra produced a weaker but detectable band of the same size. Two
nonpigmented (albino) strains of A. fumigatus and a strain
of A. fumigatus var. sclerotiorum showed faint or
no reactivity under these conditions.
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FIG. 2.
DNA from Aspergillus spp. amplified with
primers Z19-276 and Z19-660 by either a conventional annealing protocol
(A) or a touchdown protocol (B). Lanes 1 and 10, A. fumigatus CDC MAS92-229; lanes 2 and 11, A. fumigatus
NRRL 163; lanes 3 and 12, N. spinosa NRRL 5034; lanes 4 and
13, N. fennelliae NRRL-A22212; lanes 5 and 14, A. flavus MAS92-30; lanes 6 and 15, A. flavus MAS92-230;
lanes 7 and 16, A. niger MAS92-641; lanes 8 and 17, A. niger MAS92-243; lanes 9 and 18, no-template control. BP, base
pairs.
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FIG. 3.
DNA from members of Aspergillus section
Fumigati (A. fumigatus or Neosartorya
spp.) amplified by RAPD-PCR with the OPZ19 primer (A), by conventional
PCR with primers Z19-276 and Z19-660 (B), or by touchdown PCR with
primers Z19-276 and Z19-660 (C). Lanes 1, A. fumigatus var.
ellipticus NRRL 5109; lanes 2, A. fumigatus NRRL
163; lanes 3, N. spinosa NRRL 5034; lanes 4, A. fumigatus var. helvola NRRL 2244; lanes 5, N. aureola NRRL 2244; lanes 6, A. fumigatus var.
sclerotiorum NRRL 6137; lanes 7, N. fischeri NRRL
181; lanes 8, N. glabra NRRL 2163; lanes 9, N. pseudofischeri NRRL 3496; lanes 10, A. fumigatus var.
acolumnaris NRRL 5587; lanes 11, N. fischeri NRRL
A7223; lanes 12, N. fennelliae NRRL A22212; lanes 13, A. fumigatus CDC 95-11279 (albino); lanes 14, A. fumigatus CDC 95-11277 (albino); lanes 15, A. fumigatus
CDC MAS92-229; lanes 16, A. fumigatus CDC MAS92-564; lanes
17, A. fumigatus CDC B5051; lanes 18, A. fumigatus CDC 1809; lanes 19, no-template control. BP, base
pairs.
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TaqMan assay system screening of homologous and heterologous
clinical isolates.
The TaqMan assay system was adapted for
automated fluorogenic detection of the 864-bp A. fumigatus-specific amplicon and the 377-bp rRNA control amplicon
simultaneously. Seventy-one clinical isolates of A. fumigatus from San Francisco were simultaneously screened with the
Z19-derived primer pair and with the control primers NS7 and NS8 (Fig.
4 and Table
1). In order to rule out the possibility
that the 864-bp amplicon represented a geographic variant unique to San
Francisco, 29 clinical, environmental, and stock collection isolates of
A. fumigatus from a variety of additional geographic
locations were tested.
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FIG. 4.
PCR for detection of the 864-bp A. fumigatus
diagnostic product (FAM-labeled probe) and the 377-bp fungal rRNA
control (HEX-labeled probe). Lane 1, no-template control; lane 2, plasmid 19B1 (FAM probe-only control); lane 3, A. flavus CDC
MAS92-30 (HEX probe-only control); lane 4, A. fumigatus CDC
MAS92-229 (FAM-positive control); lane 5, A. fumigatus
MAS92-564 (FAM-positive control); lane 6, A. versicolor
MAS92-490 (FAM- and HEX-indeterminate control); lane 7, A. flavus MAS92-230 (FAM-negative control); lane 8, plasmid 19B1 (HEX
negative); lane 9, A. fumigatus MAS93-805; lane 10, A. terreus MAS93-997; lane 11, A. flavus MAS93-1040; lane
12, A. fumigatus var. ellipticus NRRL 5109; lane
13, A. fumigatus NRRL 163; lane 14, N. spinosa
NRRL 5034; lane 15, A. fumigatus var. helvola
NRRL 174; lane 16, N. aureola NRRL 2244; lane 17, A. fumigatus var. sclerotiorum NRRL 6137; lane 18, N. fischeri NRRL 181; lane 19, N. glabra NRRL
2163.
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TABLE 1.
Comparison of TaqMan PCR and ethidium bromide staining
for detection of the 800-bp A. fumigatus-specific amplicon in a touchdown PCR
annealing format
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DNAs from 89 isolates of
A. fumigatus were detected by the
TaqMan assay as an elevation in FAM fluorescence that correlated
with
the presence of an 864-bp band that intensely stained with
ethidium
bromide after agarose gel electrophoresis (Fig.
4 and
Table
1). These
isolates included 85 of 89 morphologically typical
clinical
A. fumigatus strains, the type strain (
A. fumigatus NRRL
163),
A. fumigatus var.
acolumnaris,
A. fumigatus var.
ellipticus,
and
A. fumigatus
var.
helvola. Data from a representative experiment
are
shown in Fig.
5. Of the remaining 11
A. fumigatus isolates,
DNA from one morphologically typical
strain produced an indeterminate
FAM result but showed the 864-bp band
on a stained gel (scored
as positive). DNAs from three morphologically
typical strains,
six white (albino) strains, and
A. fumigatus var.
sclerotiorum produced no elevated FAM
signal and no 864-bp product. When these
samples were retested at a
lower annealing temperature (56°C),
all produced both an elevated FAM
result and the 864-bp band (Fig.
3 and data not shown).
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FIG. 5.
Representative results from a TaqMan PCR experiment
performed with the templates shown in Fig. 4. Results are expressed as
normalized FAM result (A. fumigatus-specific probe) versus
normalized HEX result (rRNA control probe). Arrows indicate the
positions of the positive and negative cutoffs (0.8 and 0.35, respectively) for this experiment. Diamonds indicate TaqMan assay
results for control templates: 1, A. flavus MAS92-230 (FAM
negative, HEX positive); 2, A. versicolor MAS92-490 (FAM and
HEX indeterminate); 3, plasmid 19B1 (FAM positive, HEX negative); 4, A. fumigatus CDC MAS92-229 (FAM and HEX positive); 5, A. fumigatus CDC MAS92-564 (FAM and HEX positive).
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Neosartorya aureola,
Neosartorya spinosa,
N. fenneliae, and
Neosartorya pseudofischeri
produced no FAM reactivity and no visible
864-bp amplicon under
touchdown conditions.
N. glabra and both
isolates of
N. fischeri produced faint amounts of the 864-bp amplicon
(scored as negative), with no elevated FAM reactivity (Fig.
4).
One San
Francisco isolate originally identified as
A. fumigatus by
the referring institution was later confirmed as a
Neosartorya sp. at the CDC Fungal Reference Laboratory. This
template generated
an indeterminate FAM result, and the 864-bp amplicon
was present
(scored as positive).
Fifty-six isolates of heterologous species (47 isolates of
Aspergillus species and 9 isolates of morphologically
similar
Penicillium and
Paecilomyces species)
were included (Table
1). DNAs from
51 isolates demonstrated no
false-positive results either in the
TaqMan assay or on a stained
agarose gel. DNA from one isolate
of
A. flavus, one isolate
of
A. versicolor, and three isolates
of
A. nidulans produced an indeterminate FAM result, but no 864-bp
amplicon was detected. These results were interpreted as negative.
When
these 56 samples were retested at lower stringency (56°C),
the 864-bp
band still could not be detected.
The sensitivity of the TaqMan assay was comparable to that of ethidium
bromide staining. When a known positive template was
tested in a
dilution series, the detection limit for an unequivocal
positive result
was approximately 10 ng of input template DNA,
and 1 ng to 10 pg
correlated with an indeterminate result. With
less than 10 pg both
TaqMan assay and staining results were negative
(data not shown).
TaqMan assay results with a template concentration
of 50 ng per 50-µl
reaction volume were consistently reproducible,
so this template
concentration was selected for subsequent assays.
All tested samples demonstrated an increased HEX signal that correlated
with amplification of the 377-bp rRNA control target.
On initial
testing, two samples (one
A. versicolor isolate and
one
A. nidulans isolate) failed to generate either an elevated
HEX signal or the control band. DNA was reisolated from these
cultures,
and the control was successfully amplified from the
new preparations.
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DISCUSSION |
A variety of filamentous fungi have been increasingly recognized
as agents of serious disease in susceptible patient populations (1, 21). Accurate and timely identification of the causative agent is extremely important for the diagnosis and management of these
diseases, as well as for surveillance and epidemiologic studies
(6).
One activity of this laboratory is to explore the utility of novel
technologies potentially useful as rapid, straightforward alternatives
to traditional microscopic and phenotypic identification methods for
filamentous fungi. Nucleic acid-based identification strategies have
been developed for many pathogenic fungi (25; for a
review see reference 18), but many of these
approaches require extensive DNA sequence information,
postamplification sample manipulation, radioisotope usage, or other
advanced technical and interpretive skills. In this investigation, we
developed PCR primers to identify A. fumigatus in unknown
fungal isolates. These primers amplify a fragment originally identified
by DNA fingerprinting with arbitrary decamer primers at low stringency.
Our goal was to develop an assay that would discriminate closely
related species within the section Fumigati (23)
while simultaneously recognizing a range of morphologic variants as members of the species A. fumigatus (13). Other
investigators have attempted to achieve similar goals by using selected
rRNA gene regions as amplification targets (12, 16, 20, 25); however, taxonomically related strains had not been tested. In this
investigation, we used identical reagents in annealing formats of
differential specificities: a touchdown annealing for discrimination of
A. fumigatus and lower-stringency annealing to include
Neosartorya spp. and nonpigmented A. fumigatus
strains.
The TaqMan fluorescent assay represents a novel development in the
detection of amplified PCR products. In our laboratory, this technique
enabled samples to be analyzed rapidly (as soon as 5 to 10 min after
PCR was completed) without postamplification manipulations, which can
be technically challenging and which can provide a significant source
of laboratory contamination. Furthermore, use of the TaqMan assay
provides an additional level of diagnostic specificity, because the
fluorescent probe will not be cleaved unless it is hybridized to its
complementary target sequence.
By using touchdown annealing conditions, the Z19-derived primer-probe
combination successfully amplified and detected DNAs from 89 of 100 A. fumigatus isolates obtained from a variety of geographic
locations (including A. fumigatus var.
ellipticus, A. fumigatus var. helvola,
and A. fumigatus var. acolumnaris). The TaqMan
assay did not detect one morphologically typical A. fumigatus isolate (indeterminate TaqMan assay result), but the amplicon was detected on a stained gel. DNAs from three morphologically typical clinical isolates, six nonpigmented (albino) strains, and the
stock strain of A. fumigatus var. sclerotiorum
did not react with either the PCR primers or the corresponding TaqMan probe under these conditions. The detection sensitivity for the primer-probe combination, with gel electrophoresis used to resolve indeterminate TaqMan results, was calculated to be 90% for all 100 isolates classified as A. fumigatus or 96% if only the 94 morphologically typical isolates were included. The detection sensitivity of the TaqMan assay alone (without gel confirmation) was
89% for all isolates or 94% when the six albino strains were excluded. These reagents did not react under either touchdown or
lower-temperature (56°C) annealing conditions with any of 56 strains
of heterologous Aspergillus, Penicillium, or
Paecilomyces species, for a specificity of 100% when gel
electrophoresis was used to resolve the results for five isolates with
TaqMan assay-indeterminate results. The use of control reagents, which
coamplified and detected a 377-bp rRNA gene fragment in every sample
containing fungal DNA, identified false-negative results presumably
caused by PCR inhibition. In two cases, this situation was corrected by
producing new template DNA and repeating the test.
Neosartorya species are taxonomically most closely related
to A. fumigatus and are traditionally differentiated by
electron microscopy (23). The reagents in this study
identified Neosartorya species in a differential manner. DNA
from one of nine isolates was amplified under touchdown annealing
conditions, but it was not detected with the TaqMan probe
(indeterminate result). A faint band was detected after touchdown PCR
with several additional species; we have interpreted this as a negative
result because the amount of 864-bp target varies in replicate
experiments and consistently fails to match the abundant amounts of
amplicon produced by DNA from A. fumigatus samples under
these conditions. Furthermore, the TaqMan probe is not cleaved under
touchdown annealing conditions (indeterminate or negative TaqMan
results). Decreasing the annealing stringency to 56°C improved the
consistency of amplification so that eight of nine isolates could be
detected with these reagents. A higher percentage of nucleotide
sequence mismatches between primer and target can cause poor
reproducibility of reactions at comparatively higher annealing
temperatures and/or when competing with a more efficient annealing
reaction such as that of the 377-bp control template (Fig. 4). We are
investigating this presumed differential stability of
Neosartorya primer-template hybrids at different annealing
temperatures as a possible means of discriminating among members of the
section Fumigati and between Fumigati and members
of other Aspergillus sections. We are examining these sequences directly to investigate the extent of variation (work in
progress). Our results confirm previous molecular taxonomic evidence of
variation among A. fumigatus and Neosartorya
species. Peterson (23) showed that four strains of A. fumigatus had total DNA sequence similarity of 92 to 100%, while
the similarity between A. fumigatus and the most closely
related Neosartorya species, N. fischeri, was 65 to 68%. Girardin et al. (11) reported that a moderately
repetitive DNA probe that hybridizes efficiently with A. fumigatus DNA shows only weak reactivity with N. fischeri, N. glabra, and N. spinosa.
DNA from 10 isolates of A. fumigatus also displayed
differential reactivities with these reagents (amplification at the
lower annealing temperature but not at the higher annealing
temperature). It is interesting that 6 of the 10 strains currently
classified as A. fumigatus but nonreactive with the
Z19-derived primers at 65°C are regarded as nonpigmented (albino)
A. fumigatus variants. Possibly, this amplicon may have a
role in pigment production for strains of the section
Fumigati. It is also possible that the nonpigmented strains
may require reclassification (work in progress).
With DNA from one A. fumigatus isolate, the 864-bp target
was successfully amplified but not detected (indeterminate FAM signal in the TaqMan assay). Conversely, with five isolates of heterologous species, an indeterminate FAM signal was generated but the amplicon was
not produced. Similar results were obtained by altering the template
concentration from 1 to 100 ng, by repeat testing of the same template
DNA preparation, or by preparing another template sample. However,
indeterminate results could be resolved by examining a stained gel for
the presence of the 864-bp amplicon. Indeterminate results presumably
arise from sequence mismatch at the region of the TaqMan probe, which
could lead to poor probe binding and probe cleavage failure even when
the target could be amplified.
RAPD-PCR and the TaqMan PCR assay appear to be useful strategies for
identifying and detecting diagnostic DNA targets. Because no prior DNA
sequence information is required, RAPD-PCR allows diagnostic
information to be readily obtained even for fungal species with little
or no previous molecular characterization. Although we have validated
this technology with DNA prepared from fungal cultures, the TaqMan
assay has also been used with template DNA prepared directly from
clinical materials (19, 26). These approaches appear
promising in facilitating laboratory identification and taxonomic
classification of filamentous fungal isolates. The clinical utility of
these tools in the diagnosis and management of fungal diseases remains
to be determined.
| |
ACKNOWLEDGMENTS |
We thank S. Peterson and L. J. R. Milne for generous
gifts of Aspergillus and Neosartorya strains. We
also gratefully acknowledge the many participants in the CDC Fungal
Active Surveillance Project, who collected isolates for this study. We
thank Anne Whitney, Claudio Sacchi, and Linda Weigel for assistance
with DNA sequencing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Mycotic Diseases
Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mailstop D-11, Atlanta, GA 30333. Phone: (404) 639-2842. Fax: (404)
639-4421. E-mail: mbb4{at}cdc.gov.
| |
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Abstract
Introduction
