A simple polymerase chain reaction-sequencing analysis capable of identifying multiple medically relevant filamentous fungal species

Abstract

Due to the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces it is necessary to accurately determine the organisms responsible for these maladies and to identify them in an accurate and timely manner. Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. To this end, we have devised a simple PAN-PCR approach which when coupled to cloning and sequencing of the clones allows for the unambiguous identification of multiple fungal organisms. Universal primers are used to amplify ribosomal DNA sequences which are then cloned and transformed into Escherichia coli. Individual clones are then sequenced and individual sequences analyzed and organisms identified. Using this method we were capable of identifying Stachybotrys chartarum, Penicillium purpurogenum, Aspergillus sydowii, and Cladosporium cladosporioides from a mixed culture. This method was found to be rapid, highly specific, easy to perform, and cost effective.

Full text

A simple polymerase chain reaction-sequencing analysis capable
of identifying multiple medically relevant filamentous fungal species
Timothy R. Dean
1
, Michael Kohan
2
, Doris Betancourt
1
& Marc Y. Menetrez
1
1
National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 T.W.
Alexander Drive, Research Triangle Park, NC, 27711, USA;
2
National Health and Environmental Effects
Research Laboratory, U.S. Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle
Park, NC, 27711, USA
Received 5 April 2006; accepted in revised form 6 September 2006
Abstract
Due to the accumulating evidence that suggests that numerous unhealthy conditions in the indoor envi-
ronment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces it is
necessary to accurately determine the organisms responsible for these maladies and to identify them in an
accurate and timely manner. Historically, identification of filamentous fungal (mold) species has been based
on morphological characteristics, both macroscopic and microscopic. These methods may often be time
consuming and inaccurate, necessitating the development of identification protocols that are rapid, sen-
sitive, and precise. To this end, we have devised a simple PAN-PCR approach which when coupled to
cloning and sequencing of the clones allows for the unambiguous identification of multiple fungal
organisms. Universal primers are used to amplify ribosomal DNA sequences which are then cloned and
transformed into Escherichia coli. Individual clones are then sequenced and individual sequences analyzed
and organisms identified. Using this method we were capable of identifying Stachybotrys chartarum,
Penicillium purpurogenum,Aspergillus sydowii,andCladosporium cladosporioides from a mixed culture. This
method was found to be rapid, highly specific, easy to perform, and cost effective.
Key words: filamentous fungi, fungal identification, mold, PAN-PCR, sequencing, Stachybotrys chartarum
Introduction
In recent years there has been an increase in the
awareness of the importance of a healthy indoor
environment. A central dynamic affecting the
quality of the indoor environment is the control
and removal of biological contaminants, mainly
the filamentous fungi (mold). Estimates of fungal
contamination of homes in North America indi-
cate that up to 40% contain mold growth, while in
other parts of the world such as Northern Europe
the proportion of fungal contaminated homes
ranges between 20 and 40% [1, 2]. Fungal
contaminants that can inundate the indoor
environment include microbial volatile organic
compounds (MVOC), allergenic proteins, and in
some cases mycotoxins [3, 4, 5].
Adverse health effects that have been attributed
to the filamentous fungi include itchy eyes, stuffy
nose, fatigue, headache, and in severe cases idio-
pathic pulmonary hemosiderosis (IPH) in infants
resulting in death [6–14]. The term ‘‘sick building
syndrome’’ accurately reflects the potential that
molds can have on the built environment [15].
Currently, only a small percentage of these fungal
contaminants have been implicated in adverse
health effects, however, with the increased inter-
est and research aimed at these organisms it is
Mycopathologia (2006) 162: 265–271 Springer 2006
DOI: 10.1007/s11046-006-0068-z

probable that the list of organisms that induce ill
health will be expanded.
Fungal organisms have historically been iden-
tified based on morphological characteristics, both
macroscopic as well as microscopic. Examination
of the traits and distinctions of the colonies, and
morphological characteristics such as conidial size,
texture, shape, and structure are all commonly
used methods of identification [16–20]. These
methods may require up to two weeks for an
identification to be made making them time con-
suming and highly inaccurate. It is extremely dif-
ficult to distinguish between organisms that are
similar morphologically. Additionally, not all of
the organisms in a sample will be culturable [6].
This inevitably leads to misidentification and
understatement of the organisms that constitute
the microbial community [21]. Due to these con-
cerns it is imperative that new methods of fungal
identification be developed that are rapid, specific,
easy to perform, and cost effective.
Because of these concerns, molecular biology
techniques have been developed that circumvent
many of the issues of morphological identification.
Techniques that have proven successful include
quantitative Polymerase Chain Reaction (qPCR),
restriction fragment length polymorphism (RFLP)
analysis, random amplified polymorphic DNA
(RAPD) analysis, and image analysis. Each of these
methods has been used successfully to identify and/
or quantify fungal organisms from a number of
different environmental samples [22–27].
These methods enable rapid, sensitive, and
specific identification of fungal organisms, how-
ever in most cases they are only being used to
identify single organisms from complex environ-
mental samples. Fungal organisms found in the
environment are rarely if ever encountered singly.
A more practical approach is the identification of
numerous organisms from a single environmental
sample. Identification of multiple fungal species in
a single PCR based reaction can save time and
money, while maintaining high specificity and
accuracy. Here we describe a PAN-PCR coupled
to cloning and sequencing of the cloned insert that
we have developed and optimized. This method is
capable of identifying four organisms via analysis
of the ribosomal DNA sequence. We will also
show that this method is suitable for identification
of many fungal organisms from a single environ-
mental sample.
Materials and methods
Fungal isolates
Aspergillus sydowii,Cladosporium cladosporioides,
and Stachybotrys chartarum were all kindly pro-
vided by Research Triangle Institute (RTI). All of
the organisms provided by RTI were environ-
mental isolates obtained from environmental dust
samples from houses in Cleveland, Ohio. Penicil-
lium purpurogenum was kindly provided by Steve
Vesper from EPA/ORD/NERL. It was also iso-
lated from environmental dust samples from
houses in Cleveland, Ohio.
Growth and harvest of spores
All fungal organisms were grown on Sabouraud
Dextrose Agar (SDA) plates. Plates were prepared
according to the suppliers instructions. Each
organism was plated and grown to confluence on
three different SDA plates in preparation for spore
harvest. Organisms were allowed to grow for at
least 10 days prior to spore harvest. Spores were
harvested as previously reported [28, 29]. Spores
were harvested from plates with 3 mL of 0.01 M
phosphate buffer with 0.05% (v/v) Tween 20
(Sigma Chemical, St. Louis, MO, USA) by gently
agitating the plate surface with a bent glass rod.
The supernatant from the three plates was com-
bined and the spore suspension centrifuged at
12,000 g for 5 min. The supernatant was then
decanted leaving the spore pellet intact. The pellet
was washed three times with 10 mL of phosphate
buffer and stored at 4 C until needed. The total
spore counts were enumerated by direct micro-
scopic counting on a hemacytometer as described
by Roe [30].
Fungal DNA purification
The spore DNA was purified as previously repor-
ted [28]. The spores were mechanically broken
open using a bead milling method followed by a
phenol:CHCl
3
–ethanol precipitation step. For
bead milling 0.25 g of acid-washed glass beads
(212–311 lm) were placed in a 2 mL screw cap
conical tube. A volume of 200 ll or approximately
10
7
spores were added to the glass beads. The tube
was then shaken in a mini bead beater (Biospec
Products, Bartlesville, OK) for 50 s at the maximal
266

rate. The tube was then placed on ice for 1 min to
cool the sample and then shaken a second time.
The supernatant was removed from the beads and
subjected to a phenol:CHCl
3
extraction and an
ethanol precipitation [31]. Following precipitation
the samples were stored at )20 C until needed.
Primers and PCR conditions
PCR reactions were carried out using forward primer
ITS-1 (5¢-TCCGTAGGTGAACCTGCGG-3¢)and
reverse primer ITS-4 (5¢-TCCTCCGCTTATTGA-
TATGC-3¢) [32]. These primers are considered
universal fungal primers and have been shown to
amplify the organisms used in this study [21]. Initial
PCR optimization consisted of obtaining amplifica-
tion of each target gene under individual reaction
conditions. In the end, each PCR reaction contained:
0.2 mM each dNTP, 1.5 mM MgCl
2
,1.0lMeach
forward and reverse primers, 1.5 U Platinum Taq
DNA polymerase, Buffer (50 mM KCl, 10 mM
Tris–HCl, pH 9.0 at 25 C, 0.1% Triton X-100), and
variable template concentrations. PCR was per-
formed for 35 cycles of 96 C30s;50C15s;and
68 C 2 min. PCR products were separated by elec-
trophoresis in 2% low melting point agarose, and
visualized by ethidium bromide staining. To confirm
that the proper ribosomal sequences were being
amplified eachPCR product was sequenced using an
ABI 3100 Genetic Analyzer with the output
sequences analyzed for accuracy.
Plasmid construction and transformation
Plasmids were constructed and Escherichia coli
DH5awere transformed using the TOPO TA
Cloning system (Invitrogen Life Technologies,
Carlsbad, CA). Reactions were carried out fol-
lowing the manufacturer’s protocols. Briefly, 1 ll
of the PCR reaction was combined with 3 ll
dH
2
O, 1 ll Invitrogen salt solution, and 1 ll
TOPO vector. The constituents were gently mixed
and incubated at room temperature for 10 min.
Following incubation, 2 ll of the reaction mixture
was added to 1 vial of one shot cells for transfor-
mation. Following gentle mixing the reaction was
placed on ice for 30 min, followed by a heat shock
for 30 s at 42 C. Following heat shock, 250 llof
SOC media were added to the reaction mixture,
mixed gently and incubated at 37 C for 1 h at
200 rpm. After incubation 10 or 50 ll was plated
onto Luria-Bertani media (LB media) contain-
ing 50 lg/ kanamycin and 40 ll of 40 mg/ X-gal
in dimethyl formamide. Plates were incubated
overnight at 37 C. Following incubation white
colonies were chosen and transferred to LB broth
containing 50 lg/ kanamycin and grown overnight
for plasmid harvest. Plasmids were harvested using
the QIAprep Spin Miniprep system following the
manufacturer’s protocols (Qiagen, Inc. Valencia,
CA.).
Sequencing
Genetic sequencing of the amplified ribosomal
sequences was carried out utilizing the Big Dye
Terminator system (Applied Biosystems, Foster
City, CA). To ensure that the entire amplified
fragment was accurately sequenced, M13 forward
(5¢-GTAAAACGACGGCCAG-3¢) and reverse
(5¢-CAGGAAACAGCTATGAC-3¢) primers were
used. These primers anneal to locations on the
plasmid directly upstream and downstream of the
cloning site. Briefly, 1 ll of forward or reverse
primer was combined with 6 lldH
2
O, 5 ll plas-
mid template, 4 ll 2.5buffer, and 4 ll Big Dye
terminator ready reaction mixture and cycled
through the same PCR regimen cited above except
that only 25 cycles of replication were necessary.
Following removal of dye terminators (Micro Bio-
Spin P-30 spin columns Bio-Rad Laboratories,
Hercules, CA) samples were analyzed on an ABI
3100 genetic analyzer (Applied Biosystems, Foster
City, CA) utilizing ABI Sequencing Analysis
Software version 3.7.
Analysis on gypsum wallboard
Pieces of gypsum wallboard were cut into
coupons measuring 1.5’’ by 3.0’’ by 0.25’’. In
order to make the coupons suitable for fungal
growth each piece was wetted with 10 ml steril-
ized dH
2
O. After allowing the dH
2
O to soak into
the wallboard, 400 ll of 0.01 M phosphate buffer
with 0.05% (v/v) Tween 20 (Sigma Chemical,
St. Louis, MO, USA) containing 10
6
spores of
each Penicillium purpurogenum,Stachybotrys
chartarum,Aspergillus sydowii, and Cladosporium
cladosporioides was pipetted into the center of the
coupon. The spores were then allowed to grow
for 3 weeks at room temperature and 100 %
relative humidity.
267

Fungal material was harvested from the wall-
board coupons via wiping with a water moistened
sterile swab and placing into sterile dH
2
O. DNA
extraction and all subsequent enzymatic manipu-
lations, cloning and sequencing were completed
exactly as described as above.
Results and discussion
The rationale behind the development of this
experimental design was to develop and optimize a
fungal screen capable of identifying numerous
medically relevant indoor contaminants. The
organisms used in this study (Penicillium purpur-
ogenum,Stachybotrys chartarum,Aspergillus syd-
owii, and Cladosporium cladosporioides) were all
chosen based on their prevalence in buildings
contaminated with fungal growth [7]. Research
has shown that these organisms may possibly
serve as signature species for unhealthy indoor
environments.
Prior to PAN fungal competitive PCR each
organism was subjected to individual PCR to
ensure that sufficient amplification was obtained
with little or no spurious product formation and to
confirm that the correct sequence was being
amplified (Table 1). Primers ITS-1 and ITS-4 were
used to generate amplified ribosomal fragments.
These primers amplify from the 18S ribosomal
RNA gene, through the internal transcribed spacer
1, 5.8S ribosomal RNA gene, internal transcribed
spacer 2, and into the 28S ribosomal RNA gene.
All of the fungal strains that were used amplified
successfully producing a single PCR product of the
desired length, approximately 550–600 base pairs
(Figure 1 and Table 1). The resultant PCR prod-
ucts were very clean and did not require additional
purification prior to cloning and transformation.
Additional sequence analysis was carried out with
a thorough search of NCBI followed by alignment
and analysis with BioEdit software [33] confirming
that the proper fragments were being amplified
and that the sequences corresponded to the
organisms being used in this analysis (see accession
numbers in Table 1).
PAN-PCR reactions were carried out using the
exact conditions outlined for successful single PCR
reactions. The only variables that were adjusted
were the template concentrations and the concen-
tration of Taq polymerase. Due to the varying size
of the resultant PCR products it was sometimes
possible to qualitatively judge the presence of all
four organisms within a competitive PCR reaction
(data not shown) by comparing the individual
reaction products with the PAN-PCR products
prior to cloning and subsequent sequencing,
however, cloning and sequencing was always
necessary for positive identification.
Due to different amplification efficiencies the
template concentrations were important variables
in generating all four fragments in a single reaction
(Figure 1). A. sydowii seemed to amplify with the
greatest efficiency and required the greatest dilu-
tion down to the equivalent of 10
4
spores per
reaction. C. cladosporioides and P. purpurogenum
both generated good product amplification at 10
5
spores per reaction. S. chartarum seemed to
amplify with the least efficiency, whereby 10
6
spores per reaction were required for sufficient
product generation in the presence of the other
organisms. It is also possible that differences in
ribosomal DNA copy number impact amplifica-
tion from the different organisms allowing certain
organisms to out compete other members in the
same reaction.
Shifting from single PCR to PAN- PCR reac-
tions also required an increase in the concentration
of Taq polymerase necessary for robust product
generation (data not shown). Individual reactions
required 0.75 units of Taq polymerase for ampli-
fication, while competitive reactions required 1.5
Table 1. Competitive PCR reaction products
Fungal Isolate Accession no. PCR
Concentration
(spores)
Length
(bp)
Penicillium purpurogenum AY373926 10
5
596
Stachybotrys chartarum AY185565 10
6
581
Aspergillus sydowii AY373869 10
4
568
Cladosporium cladosporioides AY361968 10
5
551
268

units of Taq polymerase for robust product gen-
eration of all four reaction templates. This increase
is most likely due to the increase in the amount of
total template and the competition generated
between these different template copies.
Individual PCR reactions were cloned into the
TOPO TA Cloning system and subsequently
sequenced to ensure that each organism was
amenable to the procedure. Initial sequencing
reactions using either ITS-1 or ITS-4 produced
usable sequence data. However, while the middle
of the sequence read was very robust, both the 5¢
and 3¢ends of the fragment decreased in
sequencing efficiency. To compensate for the
decrease in efficiency at the ends of the sequence all
cloned products were sequenced using both the
M13 forward and reverse primers. These primers
anneal outside of the cloning site and allowed for
accurate base calls throughout the entire cloned
fragment. This increase in sequencing efficiency
allowed for unambiguous species identification to
be made with each organism based on sequence
data.
PAN competitive PCR reactions were com-
pleted and cloned into the plasmid vector. The
goal was to obtain sequence data for all four
organisms by analyzing 10% or less of the white
(transformed) colonies. Positive identification fol-
lowing analysis of only 10% of the transformed
colonies indicates the efficacy of obtaining reliable
results in a short amount of time with less cost
incurred. Table 2 clearly shows the differences that
occurred when different concentrations of initial
template were used. When each organism was
present in the competitive reaction at 10
5
spores it
was possible to identify all four fungal species
based on the resultant sequence data. How-
ever, the distribution was heavily skewed toward
A. sydowii, which was present in 11 of 18 clones.
By adjusting the initial concentrations of template
for each of the organisms it was possible to gen-
erate a fairly even distribution of clones (Table 2).
In order to test this methodology in a real
world application small pieces of gypsum wall
board were wetted with dH
2
O and inoculated with
10
6
of each Penicillium purpurogenum,Stachybot-
rys chartarum,Aspergillus sydowii, and Cladospo-
rium cladosporioides. Negative controls were
incorporated which included sterile gypsum board
that was not inoculated with fungal growth. After
allowing the fungi to grow on the wallboard for a
3 week period, it was visually observed that the
surface was completely covered with fungal
growth. Due to the morphological characteristics
of the fungal growth it was visually determined
that S. chartarum was the dominant species pres-
ent. It was at this point that the spores were har-
vested from the building material and subjected to
sequencing analysis.
We have observed in our laboratory that a
successful method of extracting fungal growth
from building materials (wallboard, ceiling tile,
etc.) is wiping or scraping with a moist sterile swab
(unpublished data). Due to the visual growth
Figure 1. Gel showing individual organism amplification as
well as differences encountered during PAN-PCR amplification
with varying amounts of templates. Lane 1: 100 bp size marker,
Lane 2: 10
6
spores Cladosporium cladosporioides, Lane 3: 10
4
spores Aspergillus sydowii, Lane 4: 10
6
spores Stachybotrys
chartarum, Lane 5: 10
5
spores Penicillium purpurogenum, Lane
6: Mix of all four organisms at above concentrations, Lane 7:
Mix of all four organisms at above concentrations except 10
5
spores Cladosporium cladosporioides.
Table 2. Number of clones generated using different fungal spore concentrations
Organism Spore Conc. No. of Clones Spore Conc. No. of Clones
C. cladosporioides 10
5
310
5
6
S. chartarum 10
5
310
6
6
P. purpurogenum 10
5
110
5
5
A. sydowii 10
5
11 10
4
2
269

characteristics noted above, following fungal
extraction from the gypsum board a microscopic
comparison was made of the spores. This com-
parison confirmed that the predominant spore
present in the mixture was Stachybotrys. This was
not a surprising result because the environmental
conditions in which the spores were allowed grow
(heavy initial wetting and 100% relative humidity)
favored Stachybotrys.
Sequencing analysis also showed that the pre-
dominant species extracted from the gypsum
board was Stachybotrys chartarum.S.chartarum
accounted for 17 of the 20 clones analyzed, while
A. sydowii accounted for two clones and C.
cladosporioides accounted for a single clone. P.
purpurogenum did not show up in the analysis. It is
believed that more natural growth conditions with
varying levels of relative humidity and wetting
would result in a more representative fungal cul-
ture, however, the identification of both A. sydowii
and C.cladosporioides, as underrepresented mem-
bers of the fungal culture, clearly shows that the
resolving power of the methodology is sufficient to
give an accurate fungal contaminant screen in the
indoor environment.
Conclusions
Our laboratory set out to develop a simple yet
effective fungal screen to identify multiple indoor
fungal contaminants. Due to the inherent difficul-
ties and inaccuracies associated with attempting to
distinguish fungal organisms based on growth and
morphological characteristics and because molec-
ular techniques that only identify single organisms
are rapidly becoming cumbersome and costly, our
laboratory set out to develop and optimize a PCR/
sequencing protocol capable of identifying multi-
ple fungal species. We accomplished this when we
were able to identify Penicillium purpurogenum,
Stachybotrys chartarum,Aspergillus sydowii, and
Cladosporium cladosporioides based on ribosomal
sequences obtained following PCR amplification
and subsequent cloning and sequencing of the
amplified fragments.
Future work extending on the above results
includes testing the procedure on increasingly
complex fungal cultures. The benefit of this pro-
tocol is the potential to identify unlimited numbers
of organisms, positive identifications being limited
by the number of clones analyzed. Additionally,
work that is being undertaken is the analysis of air
samples via PCR/sequencing. This application in
fungal screening in the indoor built environment
may extend beyond surface analysis to include air
sampling as well as surface analysis allowing for a
more complete picture of the level of contamina-
tion in an indoor space.
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Address for correspondence:Timothy R. Dean, National Risk
Management Research Laboratory, U.S. Environmental Protec-
tion Agency, 109 T.W. Alexander Drive, Research Triangle Park,
NC, 27711, USA
Phone: +919-541-2304; Fax: +919-541-2157
E-mail: dean.timothy@epa.gov
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