PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species Bele ´n Patin ˜o a , Amaia Gonza ´lez-Salgado b ,M a Teresa Gonza ´lez-Jae ´n b , Covadonga Va ´zquez a, * a Departamento de Microbiologı ´a III, Universidad Complutense de Madrid, Spain b Departamento de Gene ´tica, 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 to- wards several animal species, and has been classified by International Agency for Research on Cancer as possible carcinogen to humans (group 2B) (IARC, 1993). OTA occurs in various foodstuffs and beve- rages including a variety of cereals, beans, ground- nuts, spices, dried fruits, coffee, milk, wine and beer (Varga et al., 2001; Caban ˜es 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 Asper- gillus ochraceus group) and the section Nigri (Asper- gillus carbonarius and Aspergillus niger )(Te ´ren et 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.02.011 * Corresponding author. Department of Microbiology III, Faculty of Biology, University Complutense of Madrid, Jose ´ Antonio Nova ´is 2, 28040-Madrid, Spain. Tel.: +34 913 944 969; fax: +34 913 944 964. E-mail address: [email protected] (C. Va ´zquez). International Journal of Food Microbiology 104 (2005) 207 – 214 www.elsevier.com/locate/ijfoodmicro
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PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species
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www.elsevier.com/locate/ijfoodmicro
International Journal of Food Micro
PCR detection assays for the ochratoxin-producing Aspergillus
carbonarius and Aspergillus ochraceus species
Belen Patinoa, Amaia Gonzalez-Salgadob, Ma Teresa Gonzalez-Jaenb,
Covadonga Vazqueza,*
aDepartamento de Microbiologıa III, Universidad Complutense de Madrid, SpainbDepartamento 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 to-
wards several animal species, and has been classified
0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2005.02.011
* Corresponding author. Department of Microbiology III, Faculty
of Biology, University Complutense of Madrid, Jose Antonio
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 OCRA1-2 CAR1-2
Z.M.A.29 (a) Valladolid (Spain) A. niger + � �T.TT.A.2 (a) Zamora (Sp) A. niger + � �T.TT.A.7 (a) Zamora (Sp) A. niger + � �B.Me.A.28 (a) Leon (Sp) A. niger + � �Z.GA.A.29 (a) Valladolid (Sp) A. niger + � �CECT 2091 Canada A. niger + � �T.TT.A5 (a) Zamora (Sp) A. tubingensis � � �ZD.MF.ZD.A9 (a) Valladolid (Sp) A. tubingensis � � �T.TT.A11 (a) Zamora (Sp) A. tubingensis � � �T.MV.A.16 (a) Zamora (Sp) Aspergillus sp. � � �C.AL.A.37 (a) Zamora (Sp) Aspergillus sp. � � �T.MV.A .21 (a) Zamora (Sp) A. fumigatus + � �T.TT.A.8 (a) Zamora (Sp) A. fumigatus � � �T.TT.A.13 (a) Zamora (Sp) A. fumigatus � � �R.T.A.16 (a) Valladolid (Sp) A. fumigatus � � �CECT 2808 A. terreus � � �CECT 2907 A. awamori � � �CECT 2903 A. versicolor � � �CECT 2546 A. sclerotiorum � � �CBS 589.68a USA A. ochraceus + + �CBS 263.67a South Africa A. ochraceus + + �CBS 588.68a USA A. ochraceus + + �NRLL 3471 A. ochraceus + + �U-2003 (a) Rioja (Sp) A. ochraceus + + �CECT 2092 A. ochraceus NA + �CECT 2093 A. ochraceus NA + �CECT 2948 A. ochraceus + + �CECT 2969 A. ochraceus NA + �CECT 2970 A. ochraceus NA + �CCT 6810a (b) Brazil A. ochraceus � + �CCT 6795a (b) Brazil A. ochraceus + + �CCT 6790a (b) Brazil A. ochraceus + + �CCT 6825a (b) Brazil A. ochraceus � + �CCT 6780a (b) Brazil A. ochraceus + + �CECT 2086 A. carbonarius NA � +
242b (a) Spain A. carbonarius + � +
207b (a) Spain A. carbonarius + � +
171b (a) Spain A. carbonarius + � +
173b (a) Spain A. carbonarius + � +
178b (a) Spain A. carbonarius + � +
190b (a) Spain A. carbonarius + � +
168b (a) Spain A. carbonarius + � +
350b (a) Spain A. carbonarius + � +
325b (a) Spain A. carbonarius + � +
MUM 04.01c (a) Portugal A. carbonarius + � +
MUM 04.02c (a) Portugal A. carbonarius + � +
MUM 04.03c (a) Portugal A. carbonarius + � +
CECT 2906 P. verrucosum + � �C.AL.P.1 (a) Valladolid (Sp) P. sclerotiorum � � �
(continued on next page)
B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214 209
Table 1 (continued)
Strains Origin Species OTA OCRA1-2 CAR1-2
L (a) Rioja (Sp) P. polonicum � � �R.Te.P.4 (a) Leon (Sp) P. chrysogenum � � �B.Me.P.13 (a) Leon (Sp) P. expansum � � �CL.1 (a) Valladolid (Sp) Cladosporium sp. NA � �UCO.1 (a) Valladolid (Sp) A. consortiale NA � �T.MV.F.1 (a) Valladolid (Sp) Fusarium sp. NA � �BO.1 (a) Valladolid (Sp) Botrytis sp. NA � �(+) OTA production, (�) OTA non-production, (NA) not analysed. (a) Strains isolated from different grape varieties; (b) strains isolated from
coffee.a Strains supplied by Dr. L. Niessen (University of Munchen, Germany).b Strains supplied by Dr. V. Sanchis (University of Lleida, Spain).c Strains supplied by Dr. A. Venancio (University of Minho, Portugal).
B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214210
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 manufacturer’s 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 (Laser-
gene, Wisconsin, USA).
PCR assays were carried out using two sets of
primers: OCRA1/OCRA2 (5VCTTCCTTAGGGGTG-GCACAGC3V and 5VGTTGCTTTTCAGCGTCGGC-C3V, respectively) for A. ochraceus and CAR1/CAR2
(5VGCATCTCTGCCCCTCGG3V and 5VGGTTGGAG-TTGTCGGCAG3V, respectively) for A. carbonarius.
PCR reactions were performed in an Eppendorf
Mastercycler Gradient (Eppendorf). The PCR ampli-
fication protocol used for A. ochraceus was as fol-
lows: 1 cycle of 4 min 30 s at 95 8C, 30 cycles of 30
s at 95 8C (denaturalization), 30 s at 63 8C (anneal-
ing), 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 pg–10 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 ALof 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 (Tris–acetate 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 posi-
tion 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, re-
spectively, and the 5.8S gene was located between the
position +184 and +341.
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.
B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214 211
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. ochra-
ceus 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 per-
formed 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
1 2 3 4 5 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 1–10: CBS 589.68, CBS 263.67, NRLL