PCR identification system for the genus Aspergillus and

three major pathogenic species: Aspergillus fumigatus,

Aspergillus flavus and Aspergillus niger

CHISE SUGITA*,$, KOICHI MAKIMURA$, KATSUHISA UCHIDA$, HIDEYO YAMAGUCHI$ & ATSUSHI NAGAI*

*First Department of Internal Medicine, Tokyo Woman’s Medical University, Shinjuku, Tokyo and $Teikyo University Instituteof Medical Mycology, Hachioji, Tokyo, Japan

A PCR system was developed that allowed recognition of three major pathogenic

Aspergillus species, namely A . fumigatus, A. niger and A. flavus, in isolates

obtained from clinical specimens. The primer pair for PCR was designed from

conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA and

its flanking regions. Products 521 bp in size were successfully amplified by PCR

from the seven Aspergillus species most frequently encountered as opportunistic

pathogens, and the three most commonly significant species were identified by

separate PCR reactions or nested PCR based on use of species-specific primers. To

our knowledge, this is first report of identification of the second and third most

frequent pathogenic Aspergillus species using specific PCR amplification. The PCR

based identification system reported here will be a powerful tool to control difficult

pulmonary fungal infections and to speed the application of effective treatment.

Keywords Aspergillus species, identification, internal transcribed spacer 1

ribosomal DNA, nested PCR

Introduction

Aspergillus species have recently caused increasing

numbers of life-threatening acute invasive infections

in immunocompromized patients [1]. The steadily

increasing incidence of invasive aspergillosis over the

last few decades is ascribable to the increasing number

of patients undergoing chemotherapy, bone marrow or

solid organ transplantation and intensive corticosteroid

therapy [2].

Conventional diagnosis of fungal infection relies on

the identification of pathogens by means of morpho-

logical characters specific to the genus and species. This

is sometimes unsuccessful, however, because of the

atypical features of some isolates. Molecular biological

identification systems for pathogenic aspergilli have

been suggested as a solution to this problem: for

example, a PCR based diagnostic method for detecting

the genus Aspergillus using 18S rDNA [3,4] has been

designed. Systems have also been described for specific

detection of Aspergillus fumigatus with primers based

on regions of the 28S rDNA [5] or of the internal

transcribed spacer (ITS) 1 and 2 regions of ribosomal

DNA (rDNA) [6�/9].

These PCR systems described to date are useful only

in identifying the genus Aspergillus as a whole or the

single species A. fumigatus. The ITS region contains

variable elements that allow for sequence-based identi-

fication of Aspergillus species [10]; therefore, the region

offers a possible template for design of species-specific

primers for identification of the major pathogenic

species.Because the number of species of pathogenic fungi

known to infect immunocompromized patients is

growing [9], it is essential that quick and reliable

methods of identification be found for the most

common pathogenic species of aspergilli. This means

that not just A. fumigatus, but also Aspergillus flavus

and A. niger, should be rapidly identified by a success-

ful system.

Correspondence: K. Makimura, Teikyo University Institute of

Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan.

Tel. :�/81 426 78 3256 Fax:�/81 426 74 9190 E-mail:

makimura@main.teikyo-u.ac.jp

Received 10 January 2003; Accepted 10 November 2003

– 2004 ISHAM DOI: 10.1080/13693780310001656786

Medical Mycology October 2004, 42, 433�/437

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In the present study, we attempted to develop a rapid

and reliable identification system to identify the genus

Aspergillus in general and the three major pathogens

named. We developed an identification system based

on new primers, derived from the ITS1 region and its

flanking regions, for use in PCR and nested PCR.

Materials and methods

Organisms

Fungal strains used in this study are shown in Table 1.

Eighteen species were represented by 27 stock cultures.

Included were seven Aspergillus species represented by

16 strains, as well as two fresh clinical isolates. Clinical

strain 1 was isolated from a tumor of the lung; strain 2

was an isolate from sputum. In neither isolate could the

species be identified by its morphology because the

isolates had little or no ability to produce conidia.

DNA preparation

Strains were grown on Sabouraud dextrose agar (SDA;

peptone 1%, glucose 1%, agar 1%) at 278C for 1�/5 days

depending on strain growth rate. DNA was rapidly

prepared from strains of fungi by the method

previously described by authors of the present study

[3,11]. A small amount of fungal material grown on

SDA was placed in lysis buffer (200 mmol/l Tris-HCl

pH 8.0, 0.5% sodium dodecyl sulfate [SDS], 250 mmol/l

NaCl, 25 mmol/l ethylenediamine tetraacetic acid

[EDTA]) and, for molds, crushed with a conical

grinder. This material was then incubated at 1008Cfor 15 min and mixed with 150 ml 3.0 mol/l sodium

acetate, kept at �/208C for 10 min, then centrifuged at

10 000 g for 5 min. The supernatant was extracted once

with phenol-chloroform-isoamyl alcohol (25:24:1), and

subsequently extracted once with chloroform. DNA

was precipitated with an equal volume of isopropanol

at �/208C for 10 min, washed with 0.5 ml 70% ethanol,

dried, and suspended in 50 ml ultra pure water (Milli-Q

Table 1 Specificity of the primer sets tested in this study.

Species Strain Primer set codes

ASAP Fmi Fla Nig ITS

Aspergillus fumigatus TIMM3968 �/ �/ �/ �/ �/

2920 �/ �/ �/ �/ �/

0108 �/ �/ �/ �/ �/

A. flavus TIMM0057 �/ �/ �/ �/ �/

2935 �/ �/ �/ �/ �/

2912 �/ �/ �/ �/ �/

(A. oryzae ) 0117 �/ �/ �/ �/ �/

A. niger TIMM0113 �/ �/ �/ �/ �/

0114 �/ �/ �/ �/ �/

2915 �/ �/ �/ �/ �/

2930 �/ �/ �/ �/ �/

2932 �/ �/ �/ �/ �/

A. terreus TIMM2929 �/ �/ �/ �/ �/

A. versicolor TIMM1290 �/ �/ �/ �/ �/

A. japonicus TIMM2910 �/ �/ �/ �/ �/

A. clavatus TIMM0056 �/ �/ �/ �/ �/

Alternaria alternata TIMM3834 �/ �/ �/ �/ �/

Absidia corymbifera TIMM0002 �/ �/ �/ �/ �/

Penicillium chrysogenum TIMM0883 �/ �/ �/ �/ �/

P. expansum TIMM1293 �/ �/ �/ �/ �/

P. citreonigrum TIMM0882 �/ �/ �/ �/ �/

Pseudallescheria boydii TIMM0887 �/ �/ �/ �/ �/

Fusarium solani TIMM1304 �/ �/ �/ �/ �/

Mucor circinelloides TIMM3177 �/ �/ �/ �/ �/

Trichosporon asahii TIMM3966 �/ �/ �/ �/ �/

Cryptococcus neoformans TIMM3173 �/ �/ �/ �/ �/

Candida albicans TIMM1768 �/ �/ �/ �/ �/

ASAP, genus Aspergillus specific primer-pair; Fla, A. flavus -specific primer set; Fmi, A. fumigatus -specific primer set; ITS, general fungus

specific primer-pair; Nig, A. niger- specific primer set; TIMM, Teikyo University Institute of Medical Mycology.

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Synthesis A10; Millipore); 1 ml solution was used as the

template for PCR [11].

Oligonucleotide design

All oligonucleotides used in this study were designed

based on the relevant sequences of the ITS1 andflanking regions deposited in the DDBJ/EMBL/Gen-

Bank database (accession nos.: X78537, A. flavus ;

M55626, A. fumigatus ; X78538, A. niger ; X78540, A.

terreus ). The following two oligonucleotide primers

(termed ASAP) were designed for specific detection of

the genus Aspergillus, ASAP1:5?-CAGCGAGTACAT-

CACCTTGG-3? and ASAP2:5?-CCATTGTTGAAA-

GTTTTAACTGATT-3?. These oligonucleotides andthe others mentioned below were made by Amersham

Bioscience, Japan. The primers were expected to

amplify a fragment 521 bp in length including the

whole ITS1 region. Also, three oligonucleotide primer

sets based on specific regions within the ASAP1 and

ASAP2 amplicons were designed for use in identifying

A. fumigatus, A. niger and A. flavus. The A. fumigatus-

specific primer set (called Fmi) was made up of primersdesignated ASPU (5?-ACTACCGATTGAATGGCTC-

G-3?) and Af3r (5?-CATACTTTCAGAACAGCG-

TTCA-3?). The A. niger set (called Nig) was composed

of ASPU and Ni1r (5?-ACGCTTTCAGACAGT-

GTTCG-3?), while the A. flavus set (Fla) consisted of

ASPU and Fl2r (5?-TTCACTAGATCAGACAGAGT-

3?). Pan-fungal primers amplifying 18S rDNA regions

(B2F; 5?-ACTTTCGATGGTAGGATAG-3? and B4R;5?-TGATCGTCTTCGATCCCCTA-3? [11]) and ITS1

rDNA (18SF1; 5?-AGGTTTCCGTAGGTGAACCT-3?and 58SR1; 5?-TTCGCTGCGTTCTTCATCGA-3?[12]) were used as positive controls.

PCR

The genus specific ASAP PCR mixture contained 10 ml

10�/reaction buffer, 100 mM each of dATP, dCTP,

dGTP and dTTP, 2.5 U Taq polymerase (all Amer-

sham), 30 pmol of each primer, and 2 ml DNA template

solution. Ultra-pure water was added to increase thevolume to 100 ml. For ASAP PCR reaction, each

mixture was heated to 948C for 4 min and PCR was

performed with the following program for the thermal

cycler (GeneAmp PCR system 2400, Applied Biosys-

tems): 948C for 1 min; 558C for 2 min; and 728C for 90

s; all repeated for 30 cycles. Thermal cycling was

terminated by polymerization at 728C for 10 min.

For the species specific primer sets Fmi, Nig, andFla, PCR, 2 ml DNA solution from isolates or 1 ml of

the PCR products diluted 1/100 were used as a

template. Each mixture was heated to 948C for 4 min

and PCR was performed in cycles of 948C for 1 min;

608C for 15 s and 728C for 15 s. There were 25 cycles intotal. Thermal cycling was terminated by polymeriza-

tion at 728C for 10 min. The total cycling process

required 1.7 h.

Agarose gel electrophoresis

Between 5 and 10 ml PCR product was electrophoresed

in 1.2% agarose gel and visualized by ethidium bromide

staining and ultraviolet irradiation.

DNA sequence

PCR products were immediately sequenced by a DNA

sequencing kit (Applied Biosystems) with primers for

18S rDNA (B2F and B4R, detailed above) and an

automatic sequencer (Genetic Analyzer 310; Applied

Biosystems) used according to the manufacturer’sinstructions. The sequences were analyzed with Gene-

tyx-Mac10 software (Software Development, Tokyo,

Japan) and searched on the DDBJ/EMBL/GenBank

nucleotide database using BLAST programs.

Results

The genus-level specificity of the putative Aspergillus -specific primer system, or ASAP, was tested. A product

of 0.5 kbp was amplified by PCR from all tested

aspergilli, namely A. fumigatus, A. niger, A. flavus

(including its non-toxigenic domesticated form A.

oryzae), A. terreus, A. versicolor, A. japonicus and A.

clavatus, but not from other fungi, including Candida

albicans, Cryptococcus neoformans, Trichosporon asahii,

Mucor circinelloides, Fusarium solani, Pseudallescheria

boydii, Penicillium chrysogenum, P. citreonigrum (isolate

stored in TIMM under synonymous name P. ci-

treoviride ), P. expansum, Absidia corymbifera and

Alternaria alternata (Table 1).

The specificity of putatively species-specific primer-

sets for A. fumigatus, A. flavus and A. niger (Fmi, Fla

and Nig, respectively), was also tested. Each of these

primer-sets was successful in amplifying only the targetspecies (Table 1; Fig. 1).

Two nearly or completely nonsporulating clinical

isolates from respiratory materials, strains 1 and 2,

were subjected to the PCR identification. Strain 1 gave

a positive amplification with the 18S rDNA-based pan-

fungal primers, but it was negative by ASAP. This

means that it was not an Aspergillus species. Subse-

quent sequencing and mating analysis showed that thestrain was Schizophyllum commune (data not shown).

Strain 2 was positive by both pan-fungus PCR and

ASAP. Nested PCR showed that it was positive only

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PCR identification system for Aspergillus 435

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with primer set Fla and was negative with Fmi and Nig

(Fig. 2); therefore, it was identified as A. flavus. This

result was also confirmed by DNA sequencing (data

not shown).

Discussion

Most previous publications on PCR-based detection or

identification systems for Aspergillus spp. were based

on using 18S or 28S rDNA as target DNA. However,

the sequences in these regions are conserved across a

wide range of fungi; it is therefore difficult to design

truly species-specific primers. As reported previously,

the more variable ITS regions have proven more useful

for identification of fungal species [13].

To our knowledge, the present study is the first using

specific PCR amplification to allow identification not

just of A. fumigatus but also A. flavus and A. niger, the

second and third most frequently significant Aspergil-

lus species in opportunistic infection, using specific

PCR amplification. The importance of these species

should not be underestimated. The number of infec-

tions they cause is increasing [9,14�/16].

Species identification of moulds using histopathol-

ogy or conventional morphology is not always success-

ful. For example, a fungus ball can be produced by a

number of filamentous fungi, such as S. commune [17]

or P. boydii [18], which are able to grow saprotrophi-

cally in the human body. If these fungi grow out as

nonsporulating or otherwise atypical mycelium, identi-

fication is problematical [17]. The infections cased by

these fungi are similar to aspergillosis clinically and

pathologically; however, these fungi have poor suscept-

ibility to amphotericin B and candins, so different

antifungal drugs must be used for their control. Drug

susceptibilities, treatment procedures and prognoses

vary widely among fungal species; therefore, reliable

techniques for species identification are essential for

effective management.

The genus Aspergillus consists of about 185 species

[19]. A. fumigatus, A. niger and A. flavus account for

more than 95% of isolates causing aspergillosis [20]. In

the diagnosis of aspergillosis, identification of the

species involved is extremely important. Contamination

caused by environmental fungi poses a serious problem

and Aspergillus species, including many nonpathogens,

are ubiquitous. To identify an Aspergillus from clinical

specimens as a causal agent often requires corrobora-

tion based on repeated isolation of the same fungal

species. This must be done reliably in order to exclude

confusion arising from successive isolations of various

contaminating aspergilli.

Numerous recent reports have advocated direct

sequencing of PCR products for rapid molecular

biological identification of fungal strains. The DNA

sequence is superably reliable and informative; how-

ever, the necessary equipment is expensive and the

process takes more than 2 days. The PCR based

identification system reported here is a powerful tool

that facilitates diagnosis of infection at an early stage,

and allows prompt control of difficult pulmonary

Fig. 1 Specificity of species-specific primer sets in nested PCR.

Lanes left to right depict specific primer set Fmi for Aspergillus

fumigatus ; Fla for A. flavus ; Nig for A. niger ; ITS1, ITS1-specific

panfungal primer set used as a positive control.

Fig. 2 Identification of morphologically unidentifiable clinical

isolates using the nested PCR system. Strain 1 is a filamentous

fungus isolated from a resected pulmonary tumour, and Strain 2 is a

mould cultured from sputum. Lanes: M, molecular marker; F,

panfungal primer pair (positive control); A, primer pair ASAP

specific for Aspergillus at genus level; Fmi, A. fumigatus specific

primers as in Fig. 1; Fla, A. flavus -specific primers; Nig, A. niger -

specific primers.

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fungal infections, in part by rapid application of

effective treatment.

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