International Journal of Food Microbiology 104 (2005) 207 214

www.elsevier.com/locate/ijfoodmicro

PCR detection assays for the ochratoxin-producing Aspergillus

carbonarius and Aspergillus ochraceus species
Belen Patino a, Amaia Gonzalez-Salgado b, Ma Teresa Gonzalez-Jaen b,
Covadonga Vazquez a,*
a

Departamento de Microbiologa III, Universidad Complutense de Madrid, Spain

b
Departamento de Genetica, Universidad Complutense de Madrid, Spain

Received 6 April 2004; received in revised form 3 November 2004; accepted 5 February 2005

Abstract
Two PCR assays have been developed to detect Aspergillus carbonarius and Aspergillus ochraceus, considered the main
sources of ochratoxin A (OTA) contaminating commodities, particularly grapes, coffee and derivatives, in warm climates. The
species specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence
comparisons obtained from Aspergillus strains and have been tested in a number of strains from different origins and hosts.
These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early
detection of OTA-producing Aspergillus species and to prevent OTA entering the food chain.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Aspergillus carbonarius; Aspergillus ochraceus; Ochratoxin A; PCR; Detection; ITS

1. Introduction
Ochratoxin A (OTA) is a secondary metabolite
produced by Aspergillus and Penicillium species.
This mycotoxin has been shown to have nephrotoxic,
inmunotoxic, genotoxic and teratogenic properties towards several animal species, and has been classified
* Corresponding author. Department of Microbiology III, Faculty
of Biology, University Complutense of Madrid, Jose Antonio
Novais 2, 28040-Madrid, Spain. Tel.: +34 913 944 969; fax: +34
913 944 964.
E-mail address: covi@bio.ucm.es (C. Vazquez).
0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2005.02.011

by International Agency for Research on Cancer as

possible carcinogen to humans (group 2B) (IARC,
1993). OTA occurs in various foodstuffs and beverages including a variety of cereals, beans, groundnuts, spices, dried fruits, coffee, milk, wine and beer
(Varga et al., 2001; Cabanes et al., 2002; Petzinger
and Weidenbach, 2002; Serra et al., 2003), and its
maximum limit on several commodities for human
consumption are under legal regulation.
Two Aspergillus sections are known to produce
OTA: the section Circumdati (also called the Aspergillus ochraceus group) and the section Nigri (Aspergillus carbonarius and Aspergillus niger) (Teren et

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B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

al., 1996; Varga et al., 1996; Heenan et al., 1998).

Among the species of the section Nigri, A. carbonarius shows high ochratoxigenic potential, with
most of isolates having the ability to produce OTA
in culture (Heenan et al., 1998). It has been proposed
that A. carbonarius would be the main source of
OTA production in grapes and derivatives (Pitt,
2000; Cabanes et al., 2002) particularly in Mediterranean region (Serra et al., 2003), while A. ochraceus would be the main source of OTA in coffee
(Logrieco et al., 2003; Taniwaki et al., 2003).
Traditional diagnostic methods used in food mycology are based on macroscopic and microscopic
features and culture in appropriate media. The development of fruiting structures requires 2 to 10 days
of culture on different media (Raper and Fennell,
1965), increasing considerably the time of analysis.
On the other hand, these methods have low degree
of sensitivity, are difficult to standardize (Raper and
Fennell, 1965; Zhao et al., 2001) and misidentification can occur because some fungi may be poorly
characterised or because considerable expertise is
required.
PCR-based methods that target DNA are considered a good alternative for rapid diagnosis because of
their high specificity and sensitivity (Accensi et al.,
1999; Perrone et al., 2004; Rath and Ansorg, 2000;
Schmidt et al., 2003), especially when multi-copy
sequences are used to develop species specific primers
(Bluhm et al., 2002). ITS (internal transcribed spacer)
and IGS (intergenic spacer) regions of rDNA units are
present at 100 to 300 copies per haploid fungal genome and are considered high variable regions. The
high variability provided by these regions is particularly useful when it is necessary to discriminate
among closely related species or at intraspecific
level. Both, ITS and IGS regions, have been used to
carry out phylogenetic and population studies in filamentous fungi (Henry et al., 2000; Mirete et al., 2003,
2004; Parenicova et al., 2001; Varga et al., 2004; Zhao
et al., 2001) and to develop specific PCR assays to
identify important mycotoxigenic species affecting
commodities such as Fusarium or Aspergillus (Gonzalez-Jaen et al., 2004; Patino et al., 2004; Zhao et al.,
2001).
The objectives of this work were to develop specific primers, based on sequence information of the
ITS region, and the corresponding PCR assays to

detect the main ochratoxigenic Aspergillus species:

A. ochraceus and A. carbonarius.

2. Materials and methods

2.1. Fungal isolates and culture conditions
All the isolates used in this study, along with their
sources, are given in Table 1. The isolates come from
different sources: the Spanish Type Culture Collection
(Spain), ARS Culture Collection (USA), the Centralbureau voor Schimmel Cultures (The Netherlands)
and isolates kindly provided by Dr. V. Sanchis (University of Lleida, Spain), Dr. A. Venancio (University
of Minho, Portugal) and Dr. L. Niessen (University of
Munchen, Germany). The rest of the strains were
isolated from grapes in our laboratory. Several isolates
of species other than Aspergillus were also included in
our analysis.
Table 1 also indicates those isolates able to produce ochratoxins. Ochratoxin production was analysed according to Saez et al. (2004) by Dr. M.
Jimenez (University of Valencia). Cultures were
maintained on potato dextrose-agar (PDA, Scharlau
Chemie, Barcelona, Spain) medium at 4 8C and
stored as spore suspension in 15% glycerol at
80 8C. The isolates were cultured in 100 mL
Erlenmeyer flasks containing 20 mL liquid medium
Sabouraud (Scharlau Chemie, Barcelona, Spain).
Cultures were inoculated with mycelial disks cut
from the margins of 7-day-old colonies and incubated at 25 8C under static conditions. Mycelia
from 6-day-old cultures were harvested by filtration
through Whatman paper no. 1 and kept at 80 8C
for DNA isolation.
2.2. DNA extraction and PCR amplification
Genomic DNA of the strains was obtained using the
genomic DNA Extraction Kit (Genomix, Talent,
Trieste, Italy) following the manufacturers instructions.
All genomic DNAs used in this work were tested
for suitability for PCR amplification using primers
ITS1 and ITS4 (White et al., 1990), which amplify
the ITS region in Aspergillus. The PCR reaction was
performed in an Eppendorf Mastercycler Gradient
(Eppendorf, Hamburg, Germany) using between 10

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

209

Table 1
Strains analysed indicating, origin, species (Aspergillus sp., A. niger, A. tubingensis, A. fumigatus, A. ochraceus, A. carbonarius, A. awamori,
A. versicolor, A. sclerotiorum, Penicillium verrucosum, P. sclerotiorum, P. polonicum, P. chrysogenum, P. expansum, Cladosporium sp.,
Alternaria consortiale, Fusarium sp., Botrytis sp.), ability to produce OTA and the occurrence of PCR amplification product with the two pair
of primers: CAR1-2 and OCRA1-2
Strains

Origin

Species

OTA

Z.M.A.29 (a)
T.TT.A.2 (a)
T.TT.A.7 (a)
B.Me.A.28 (a)
Z.GA.A.29 (a)
CECT 2091
T.TT.A5 (a)
ZD.MF.ZD.A9 (a)
T.TT.A11 (a)
T.MV.A.16 (a)
C.AL.A.37 (a)
T.MV.A .21 (a)
T.TT.A.8 (a)
T.TT.A.13 (a)
R.T.A.16 (a)
CECT 2808
CECT 2907
CECT 2903
CECT 2546
CBS 589.68a
CBS 263.67a
CBS 588.68a
NRLL 3471
U-2003 (a)
CECT 2092
CECT 2093
CECT 2948
CECT 2969
CECT 2970
CCT 6810a (b)
CCT 6795a (b)
CCT 6790a (b)
CCT 6825a (b)
CCT 6780a (b)
CECT 2086
242b (a)
207b (a)
171b (a)
173b (a)
178b (a)
190b (a)
168b (a)
350b (a)
325b (a)
MUM 04.01c (a)
MUM 04.02c (a)
MUM 04.03c (a)
CECT 2906
C.AL.P.1 (a)

Valladolid (Spain)
Zamora (Sp)
Zamora (Sp)
Leon (Sp)
Valladolid (Sp)
Canada
Zamora (Sp)
Valladolid (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Valladolid (Sp)

A. niger
A. niger
A. niger
A. niger
A. niger
A. niger
A. tubingensis
A. tubingensis
A. tubingensis
Aspergillus sp.
Aspergillus sp.
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. terreus
A. awamori
A. versicolor
A. sclerotiorum
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. ochraceus
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
A. carbonarius
P. verrucosum
P. sclerotiorum

+
+
+
+
+
+

USA
South Africa
USA
Rioja (Sp)

Brazil
Brazil
Brazil
Brazil
Brazil
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Portugal
Portugal
Portugal
Valladolid (Sp)

OCRA1-2

CAR1-2

+
+
+
+
+
NA
NA
+
NA
NA
+
+
+
NA
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

(continued on next page)

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B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

Table 1 (continued)
Strains

Origin

Species

OTA

L (a)
R.Te.P.4 (a)
B.Me.P.13 (a)
CL.1 (a)
UCO.1 (a)
T.MV.F.1 (a)
BO.1 (a)

Rioja (Sp)
Leon (Sp)
Leon (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)

P. polonicum
P. chrysogenum
P. expansum
Cladosporium sp.
A. consortiale
Fusarium sp.
Botrytis sp.

NA
NA
NA
NA

(+) OTA production, (

coffee.
a
Strains supplied by
b
Strains supplied by
c
Strains supplied by

OCRA1-2

CAR1-2

) OTA non-production, (NA) not analysed. (a) Strains isolated from different grape varieties; (b) strains isolated from
Dr. L. Niessen (University of Munchen, Germany).
Dr. V. Sanchis (University of Lleida, Spain).
Dr. A. Venancio (University of Minho, Portugal).

pg and 10 ng of genomic DNA. The amplification

program used was described by Henry et al. (2000).
The amplification products were isolated by the High
Pure PCR Product Purification Kit (Roche, Germany)
and were sequenced using the ABI PRISM DNA
Sequencer (Applied Biosystems, Foster City, USA)
according to the manufacturers instructions in the
Genomic Unit of the University Complutense of
Madrid (Spain). All the strains were sequenced in
both directions. Sequences were analysed and aligned
by Clustal method using the program DNAstar (Lasergene, Wisconsin, USA).
PCR assays were carried out using two sets of
primers: OCRA1/OCRA2 (5VCTTCCTTAGGGGTGGCACAGC3V and 5VGTTGCTTTTCAGCGTCGGCC3V, respectively) for A. ochraceus and CAR1/CAR2
(5VGCATCTCTGCCCCTCGG3V and 5VGGTTGGAGTTGTCGGCAG3V, respectively) for A. carbonarius.
PCR reactions were performed in an Eppendorf
Mastercycler Gradient (Eppendorf). The PCR amplification protocol used for A. ochraceus was as follows: 1 cycle of 4 min 30 s at 95 8C, 30 cycles of 30
s at 95 8C (denaturalization), 30 s at 63 8C (annealing), 1 min at 72 8C (extension) and finally 1 cycle
of 3 min at 72 8C. In the case of A. carbonarius, the
PCR program was: 1 cycle of 4 min 30 s at 95 8C,
25 cycles of 30 s at 95 8C (denaturalization), 25 s at
59 8C (annealing), 40 s at 72 8C (extension) and
finally 1 cycle of 5 min at 72 8C. In both case,
amplification reactions were carried out in volumes
of 25 AL containing 3 AL (10 pg10 ng) of template
DNA, 1.25 AL of each primer (20 AM), 2.5 AL of
10 PCR buffer, 1 AL of MgCl2 (50 mM), 0.25 AL
of dNTPs (100 mM) and 0.2 AL of Taq DNA

polymerase (5 U/AL) supplied by the manufacturer

(Ecogen, Barcelona, Spain). PCR products were
detected in 2% agarose ethidium bromide gels in
TAE 1 buffer (Trisacetate 40 mM and EDTA
1.0 mM). The DNA ladder bReal escala no. 2Q
(Durviz, Valencia, Spain) was used as molecular
size marker.

3. Results
Table 1 shows the isolates analysed in this work
and their ability to produce OTA.
The ITS1-5.8S-ITS2 sequences of several isolates
of A. ochraceus, A. carbonarius, A. niger and other
related Aspergillus species were obtained and aligned
together with other sequences of Aspergillus species
available in the GenBank. Fig. 1 shows the alignment
of ITS1-5.8S-ITS2 sequence in three representative
strains of A. carbonarius (CECT 2086), A ochraceus
(CECT 2092) and A. niger (CECT 2091). The position of the primers and 5.8 gene are located using as
reference the beginning of ITS1 from each isolate.
Two pairs of specific primers, OCRA1/OCRA2 and
CAR1/CAR2, were designed on the basis of the
alignment of the sequences above mentioned. In A.
ochraceus, the primer OCRA1 was located within the
ITS1-rDNA at the position + 76 and the primer
OCRA2 at the position + 462 (within the ITS2), and
the 5.8S gene was located between the positions + 168
and +325. In A. carbonarius, primers CAR1 and
CAR2 were located at positions +91 and +480, respectively, and the 5.8S gene was located between the
position +184 and +341.

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

211

Fig. 1. Alignment of ITS1-5.8S-ITS2 sequence in three representatives strains of A. carbonarius (CECT 2086), A. ochraceus (CECT 2092) and
A. niger (CECT 2091) and the location of primers OCRA1/OCRA2 (underlined) and CAR1/CAR2 (bold). A dash represents the same
nucleotide. An empty space indicates a missing nucleotide.

All the Aspergillus, strains listed in Table 1 were

tested for amplification using the primer pair OCRA1
and OCRA2. A single fragment of about 400 bp was
only amplified when genomic DNA from A. ochraceus strains was used. No product was observed with
genomic DNA from the Aspergillus isolates other than
A. ochraceus nor in the case of other genera (Fig. 2).
Control amplifications of the genomic DNA with

primers ITS1 and ITS2 were positive for all the strains
analysed.
Similarly PCR amplifications of genomic DNA
from all the strains indicated in Table 1 were performed using the primers CAR1/CAR2. A single
fragment of about 420 bp was only obtained when
genomic DNA from A. carbonarius strains was used.
No amplification product was detected with DNA

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B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

3 4

6 7

8 9 10 11 M 12 13 14 15 16 17 18 19

Fig. 2. PCR amplification using primers OCRA1/OCRA2 and DNA from A. ochraceus strains, lanes 110: CBS 589.68, CBS 263.67, NRLL
3741, U-2003, CECT 2092, CECT 2948, CECT 2970, CCT 6810, CCT 6795, CCT 6825; lane 11: non-template control; lanes 1214: A. niger
(T.TT.A2, T.TT. A7 and Z.GA.A29, respectively); lanes 1516: A. carbonarius (171 and MUM 04.01, respectively); lane 17: P. verrucosum
(CECT 2906); lane 18: Cladosporium sp. (CL.1); and lane 19: A. consortiale (UCO.1). M: DNA marker.

samples from the Aspergillus isolates other than A.

carbonarius nor in the case of other genera (Fig. 3).

4. Discussion
Specific PCR assays have been developed in this
study for detection of both A. carbonarius and A.
ochraceus species, the main source of OTA contamination of food and feed products in warm climates
(WHO Report, 2002). The two sets of specific primers
used in both PCR assays have been designed on the
basis of ITS sequence comparisons of several strains
of Aspergillus species (Fig. 1), and their specificity
have been tested on a number of Aspergillus, Penicillium, Cladosporium, Botrytis and Alternaria strains
commonly associated with grapes, cereals and coffee
1

(Table 1, Figs. 2 and 3). The diverse geographical

locations and origins of the A. ochraceus and A.
carbonarius strains analysed in this work can be
considered representative of the variability of these
species. The comparison of ITS sequences of A.
ochraceus strains revealed little variability, according
to the results of an extensive study of this species
using AFLPs, which found a rather close grouping of
the A. ochraceus strains (Schmidt et al., 2003). In the
case of A. carbonarius, there are not extensive studies
available, but the ITS sequence comparison performed
in this work also revealed high similarity within the
group of A. carbonarius isolates and a clear discrimination from the diverse A. niger strains analysed
(data not shown).
The PCR assays described in this work represent
an advantage in terms of time of analysis and speci-

8 9 10 11 M 12 13 14 15 16 17 18 19

Fig. 3. PCR amplification using primers CAR1/CAR2 and DNA from A. carbonarius strains, lanes 110: CECT 2086, 242, 207, 171, 173, 178,
190, 168, MUM 04.01, MUM 04.02; lane 11: non-template control; lanes 1214: A. niger (T.TT.A2, T.TT. A7 and Z.GA.A29, respectively);
lanes 1516: A. ochraceus (U. 2003 and CECT 2092, respectively); lane 17: P. verrucosum (CECT 2906); lane 18: Cladosporium sp. (CL.1);
and lane 19: A. consortiale (UCO.1). M: DNA marker.

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207214

ficity in comparison with the conventional methods of

identifications and the more laborious molecular
methods based on AFLP profiles (Schmidt et al.,
2003), SSCP of the PCR-IGS (Rath and Ansorg,
2000), the PCR-RFLPs patterns of the ITS region
(Accensi et al., 1999) or the secondary metabolite
profiles (Parenicova et al., 2001) reported so far for
A. ochraceus or A. carbonarius.
Detection limit of ITS amplification product, defined as the clearly visible product on agarose gels
containing ethidium bromide, has been estimated between 1 and 10 pg of DNA template in Fusarium
(Bluhm et al., 2002). We found similar detection
levels with both set of primers when serial dilutions
of genomic DNA of A. carbonarius and A. ochraceus
were used as templates for PCR amplification (data
not shown). The sensitivity of our PCR assay based
on ITS sequences was, therefore, more sensitive than
primers based on single copy gene, estimated between
0.1 and 1 ng of DNA template per reaction (Bluhm et
al., 2002).
The specificity and high degree of sensitivity of the
PCR detection assays developed for A. ochraceus and
A. carbonarius provide a good tool for early detection
of these OTA-producing fungi in raw cultures such as
grapes or coffee and to prevent OTA entering the food
chain. Detection of these fungi, in the case of grapes,
it is particularly critical around harvest time, when
contamination levels and OTA production is considered high (Serra et al., 2003).

Acknowledgements
This work was supported by the Spanish MCyT
(AGL2001/2974/C05/05) and by an UCM-DANONE
project (PR248/02-11708). We wish to thank Gema
Rodrguez for skillful technical assistance.

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