PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species

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

Novais 2, 28040-Madrid, Spain. Tel.: +34 913 944 969; fax: +34

913 944 964.

E-mail address: [email protected] (C. Vazquez).

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; 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 Asper-

gillus ochraceus group) and the section Nigri (Asper-

gillus carbonarius and Aspergillus niger) (Teren et

biology 104 (2005) 207–214

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214208

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

Among the species of the section Nigri, A. carbo-

narius 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 Mediter-

ranean region (Serra et al., 2003), while A. ochra-

ceus would be the main source of OTA in coffee

(Logrieco et al., 2003; Taniwaki et al., 2003).

Traditional diagnostic methods used in food my-

cology are based on macroscopic and microscopic

features and culture in appropriate media. The de-

velopment 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 misidentifica-

tion can occur because some fungi may be poorly

characterised or because considerable expertise is

required.

PCR-based methods that target DNA are consid-

ered 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 ge-

nome and are considered high variable regions. The

high variability provided by these regions is particu-

larly 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 fila-

mentous 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 (Gon-

zalez-Jaen et al., 2004; Patino et al., 2004; Zhao et al.,

2001).

The objectives of this work were to develop spe-

cific 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 Central-

bureau voor Schimmel Cultures (The Netherlands)

and isolates kindly provided by Dr. V. Sanchis (Uni-

versity 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 pro-

duce ochratoxins. Ochratoxin production was ana-

lysed 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 incu-

bated 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 8Cfor 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 manufacturer’s 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

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 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

3741, U-2003, CECT 2092, CECT 2948, CECT 2970, CCT 6810, CCT 6795, CCT 6825; lane 11: non-template control; lanes 12–14: A. niger

(T.TT.A2, T.TT. A7 and Z.GA.A29, respectively); lanes 15–16: 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.

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214212

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 contam-

ination 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, Penicil-

lium, Cladosporium, Botrytis and Alternaria strains

commonly associated with grapes, cereals and coffee

1 2 3 4 5 6 7 8 9 10 11

Fig. 3. PCR amplification using primers CAR1/CAR2 and DNA from A. ca

190, 168, MUM 04.01, MUM 04.02; lane 11: non-template control; lanes

lanes 15–16: A. ochraceus (U. 2003 and CECT 2092, respectively); lane 1

and lane 19: A. consortiale (UCO.1). M: DNA marker.

(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 discrim-

ination 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-

M 12 13 14 15 16 17 18 19

rbonarius strains, lanes 1–10: CECT 2086, 242, 207, 171, 173, 178,

12–14: A. niger (T.TT.A2, T.TT. A7 and Z.GA.A29, respectively);

7: P. verrucosum (CECT 2906); lane 18: Cladosporium sp. (CL.1);

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214 213

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, de-

fined as the clearly visible product on agarose gels

containing ethidium bromide, has been estimated be-

tween 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 consid-

ered 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

Rodrıguez for skillful technical assistance.

References

Accensi, F., Cano, J., Figuera, L., Abarca, M.L., Cabanes, F.J.,

1999. New PCR method to differentiate species in the Asper-

gillus niger aggregate. FEMS Microbiology Letters 180,

191–196.

Bluhm, B.H., Flaherty, J.E., Cousin, M.A., Woloshuk, C.P., 2002.

Multiplex polymerase chain reaction assay for the differential

detection of trichothecene- and fumonisin-producing species

of Fusarium in cornmeal. Journal of Food Protection 65,

1955–1961.

Cabanes, F.J., Accensi, F., Bragulat, M.L., Abarca, M.L., Castella,

G., Minguez, S., Pons, A., 2002. What is the source of ochra-

toxin A in wine? International Journal of Food Microbiology 79,

213–215.

Gonzalez-Jaen, M.T., Mirete, S., Patino, B., Lopez-Errasquın, E.,

Vazquez, C., 2004. Genetic markers for the analysis of variabil-

ity and for production of specific diagnostic sequences in fumo-

nisin-producing strains of F. verticillioides. European Journal of

Plant Pathology 110 (5–6), 525–532.

Heenan, C.N., Shaw, K.J., Pitt, J.I., 1998. Ochratoxin A produc-

tion by Aspergillus carbonarius and A. niger isolates and

detection using coconut cream agar. Journal of Food Mycology

1, 67–72.

Henry, T., Iwen, P.C., Hinrichs, S.H., 2000. Identification of Asper-

gillus species using internal transcribed spacer regions 1 and 2.

Journal of Clinical Microbiology 38, 510–1515.

International Agency for Research on Cancer (IARC), 1993. Some

Naturally Occurring Substances: Food Items and Constituents,

Heterocyclic Aromatic Amines and Mycotoxins, vol. 56. World

Health Organization, Lyon, France.

Logrieco, A., Bottalico, A., Mule, G., Moretti, A., Perrone, G.,

2003. Epidemiology of toxigenic fungi and their associated

mycotoxins for some Mediterranean crops. European Journal

of Plant Pathology 109, 645–667.

Mirete, S., Patino, B., Vazquez, C., Jimenez, M., Hinojo, M.J.,

Soldevilla, C., Gonzalez-Jaen, M.T., 2003. Fumonisin produc-

tion by Gibberella fujikuroi strains from Pinus species. Inter-

national Journal of Food Microbiology 89, 213–221.

Mirete, S., Vazquez, C., Mule, G., Jurado, M., Gonzalez-Jaen, M.T.,

2004. Differentiation of Fusarium verticillioides from banana

fruits by IGS and EF-1a sequence analyses. European Journal

of Plant Pathology 110 (5–6), 515–523.

Parenicova, L., Skouboe, P., Frisvad, J., Samson, R., Rossen, L.,

Hoor-Suykerbyuk, M.T., Visser, J., 2001. Combined molecular

and biochemical approach identifies Aspergillus japonicus and

Aspergillus aculeatus as two species. Applied and Environmen-

tal Microbiology 67, 521–527.

Patino, B., Mirete, S., Gonzalez-Jaen, M.T., Mule, G., Rodrıguez,

T., Vazquez, C., 2004. PCR detection assay of fumonisin-pro-

ducing Fusarium verticillioides strains. Journal of Food Protec-

tion 67 (6), 1278–1283.

Perrone, G., Susca, A., Stea, G., Mule, G., 2004. PCR assay for

identification of Aspergillus carbonarius and Aspergillus japo-

nicus. European Journal of Plant Pathology 110, 641–649.

Petzinger, E., Weidenbach, A., 2002. Mycotoxins in the food

chain: the role of ochratoxins. Livestock Production Science

76, 245–250.

Pitt, J.L., 2000. Toxigenic fungi: which are important? Medical

Mycology 38, 17–22.

Raper, K.B., Fennell, D.I., 1965. The Genus Aspergillus. Williams

and Wilkins, Baltimore, MD.

Rath, P.M., Ansorg, R., 2000. Identification of medically important

Aspergillus species by a single strand conformational polymor-

phism (SSCP) of the PCR amplified intergenic spacer region.

Mycoses 43, 381–386.

B. Patino et al. / International Journal of Food Microbiology 104 (2005) 207–214214

Saez, J.M., Medina, A., Gimeno-Adelantado, J.V., Mateo, R.,

Jimenez, M., 2004. Comparison of different sample treatments

for the analysis of ochratoxin A in must, wine and beer by

liquid chromatography. Journal of Chromatography, A 1029,

125–133.

Schmidt, H., Ehrmann, M., Vogel, R.F., Taniwaki, M.H., Niessen,

L., 2003. Molecular typing of Aspergillus ochraceus and con-

struction of species specific SCAR-primers based on AFLPs.

Systematic and Applied Microbiology 26, 138–146.

Serra, R., Abrunhosa, L., Kozakiewicz, Z., Venancio, A., 2003.

Black Aspergillus species as ochratoxin A producers in Portu-

guese wine grapes. International Journal of Food Microbiology

88, 63–68.

Taniwaki, M.H., Pitt, J.I., Teixeira, A.A., Lamanaka, B.T., 2003.

The source of ocratoxin A in Brazilian coffee and its formation

in relation to processing methods. International Journal of Food

Microbiology 82, 173–179.

Teren, J., Varga, J., Hamari, Z., Rinyu, E., Kevei, F., 1996. Inmu-

nochemical detection of ochratoxin A in black Aspergillus

strains. Mycopathologia 134, 171–176.

Varga, J., Kevei, E., Rinyu, E., Teren, J., Kozakiewicz, Z., 1996.

Ochratoxin production by Aspergillus species. Applied and

Environmental Microbiology 62, 4461–4464.

Varga, J., Rigo, K., Teren, J., Mesterhazy, A., 2001. Recent

advances in ochratoxin research: I. Production, detection and

occurrence of ochratoxins. Cereal Research Communications

29, 85–92.

Varga, J., Juhasz, A., Kevei, F., Kozakiewicz, Z., 2004. Molecular

diversity of agriculturally important Aspergillus species. Euro-

pean Journal of Plant Pathology 110, 627–640.

White, T.J., Burns, T., Lee, S., Taylor, J.W., 1990. Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In: Innis, M.A., Gelgard, D.H., Sninsky, J.J.,

White, T.J. (Eds.), PCR Protocols: A Guide to Methods and

Applications. Academic Press, New York, pp. 315–322.

Who: Report of the 56th Meeting of the joint FAO/WHO Expert

Committee on Food Additives, Geneva Switzerland, 6–15 Feb-

ruary, 2002.

Zhao, J., Kong, F., Li, R., Wang, X., Wan, Z., Wang, D., 2001.

Identification of Aspergillus fumigatus and related species by

nested PCR targeting ribosomal DNA internal transcribed spac-

er regions. Journal of Clinical Microbiology 39, 2261–2266.