An overview on molecular markers for detection of ochratoxigenic fungi in coffee neans

REVIEW

An overview on molecular markers for detection of ochratoxigenic fungi in coffee beans

Maria Helena Pelegrinelli Fungaro^*; Daniele Sartori

Centro de Ciências Biológicas; Universidade Estadual de Londrina; C.P: 6001; 86055-900; Londrina - PR - Brasil

ABSTRACT

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins. Its occurrence in several agricultural commodities has been considered a worldwide problem. This toxin is mainly produced by Aspergillus species. OTA has nephrotoxic, immunosuppressive, and carcinogenic effects and consequently the contamination with this toxin represents a high risk for human and animal health. In the last 5 years, several investigators have applied molecular methods in order to develop PCR assays for identifying and quantifying OTA-producing fungi in coffee beans samples. The main objective is to allow the detection of microorganisms capable of producing OTA, preferentially prior to ochratoxin production and accumulation. In this contribution several of these attempts will be reviewed and discussed.

Key words: ochratoxigenic fungi, molecular marker, PCR detection, molecular identification

INTRODUCTION

Mycotoxins are small organic molecules with grat diversity in chemical structure and biological activity. They are toxic compounds, occasionally very hazardous to animals and human. The main source of mycotoxins is plant food, which are contaminated during harvesting, transport, storage, and manufacture, or even in the field (Smith and Henderson, 1991). Their influences on human and animal health range from neurotoxic, teratogenic, immunosuppressive, nephrotoxic, hepatotoxic to carcinogenic effects (Geisen, 1998). About 20 different mycotoxins are significant to human health (Geisen, 1998; Bennett and Klich, 2003). Demand still exists for rapid and reliable techniques to detect mycotoxins and mycotoxin-producers (Russell and Paterson, 2006).

Ochratoxin A (OTA) is a mycotoxin that is receiving increasing attention worldwide because of its severe nephrotoxicity. Moreover, the International Agency for Research on Cancer has classified OTA as a Group B2, i.e. the metabolite is a probable human carcinogen. As reviewed by Petzinger and Weidenbach (2002), OTA-contaminated foods are abundantly found. Examples are grains, coffee beans, spices, nuts, grapes, and figs (Bayman et al., 2002; Jorgensen and Jacobsen, 2002; Battilani et al., 2003; Taniwaki et al., 2003). OTA is not totally decomposed during most food processing stages such as cooking, washing, and fermenting. Because OTA is not totally decomposed during most processing stages it has been also detected in manufactured food products, such as bread, beers, wine, coffee, and chocolate (Jorgensen et al., 1998; Visconti et al., 2001; (Fujii et al., 2007). Based on its possible effects on animal and human health, most countries have imposed statutory limits for OTA in some foods. For both roasted coffee beans and ground coffee beans the maximum tolerable level of OTA in the European Community is 5μg kg⁻¹ (Official Journal of The European Union, 2005). Until 2004, the major fungal source of OTA in coffee was Aspergillus ochraceus and A. carbonarius, with minor contribution from A. niger. After advances in molecular and fungal metabolite techniques, new species were described and distinguished in the section Circumdati and Nigri. Nowadays, A. westerdijkiae is being recognized as the main OTA producer especially in arabica coffee, while A. carbonarius occurs most frequently in robusta coffee (Taniwaki et al., 2008).

The severe consequences of OTA contamination argue for methodologies that can be efficiently use to detect OTA producers in food. In this review some of the relevant molecular approaches, which have been carried out in order to detect and quantify the main OTA producers in coffee beans will be present.

Molecular Detection of Ochratoxigenic Fungi

The conventional scheme of isolation and identification of ochratoxigenic fungi from food samples are time-consuming and require high knowledge on fungal taxonomy. Even with taxonomical expertise, identification is commonly difficult regarding some fungi genus that contains a large number of closely related species. The application of molecular biology techniques can help to overcome these problems, because it can reduce the time for from several days to some hours and it allows precise species identification.

The polymerase chain reaction (PCR) assay has allowed precise identification and fast detection of ochratoxigenic species without the need for isolating pure cultures.

In contrast to others mycotoxin, the OTA biosynthetic pathway was not well characterized in any of the OTA-producing species and consequently the genes that encode enzymes involved in the biosynthesis of this secondary metabolite are poor known. Because of this, several PCR-based assays developed during the last five years have been used genes which were not associated to mycotoxin biosynthesis, as those based on ribosomal RNA, b-tubulin, and calmodulin genes (Perrone et al., 2004; Patiño et al., 2005; Morello et al., 2007) or have been based on anonymous DNA sequences. Anonymous DNA sequences are obtained from an unbiased sampling of genomic DNA and these may or may not contain functional genes. Developing markers from anonymous sequences requires comparative analyses among related species of DNA profiles generated from randomly amplified fragments by using RAPD (Random Amplified Polymorphic DNA) or AFLP (Amplified Fragment Length Polymorphism) (Williams et al., 1990; Vos et al., 1995).

Ochratoxin A was discovered as a secondary metabolite of A. ochraceus strains, which belongs to Aspergillus section Circumdati. Until recently, this species was considered the main fungal source of OTA in coffee. However, based on polyphasic taxonomy, Frisvad et al. (2004) proposed the division of formal A. ochraceus species into two species, A. ochraceus and A. westerdijkiae. These species are very similar and several isolates previously identified as A. ochraceus are now recognized as A. westerdijkiae, including the original OTA-producing strain (NRRL 3174). The first report of a diagnostic PCR assay for ochratoxigenic fungi dates from 2003 (Schmidt et al., 2003). The authors investigated the genetic relatedness among 70 A. ochraceus strains by using AFLP markers. A certain number of AFLP bands distinctive for A. ochraceus were detected. Three of these bands were cloned and sequenced and after that the sequences were used to design three primer-pairs specific for A. ochraceus. The specificity of the primer-pair OCA-V/OCA-R was tested with DNA of several different target strains as well as closely related Aspergillus and Penicillium spp. and DNA isolated from non-infected green coffee. Currently we know that this primer-pair is able to amplify DNA sequence from A. ochraceus and A. westerdijkiae, because it was developed previous to the division of formal A. ochraceus species into the two species above mentioned. Research in our group found that A. westerdijkiae is the main OTA producing species found in Brazilian coffee beans (Coffea arabica) (Fungaro et al., 2004b; Morello et al., 2007). Amplification and sequencing of the ITS1-5.8S-ITS2 region from several Brazilian strains of both species showed specific nucleotide variations (ITS and β-tubulin genes) characterizing A. westerdijkiae and A. ochraceus (Fungaro et al., 2004a; Fungaro et al., 2004b; Morello et al., 2007). The nucleotide variations found between the β-tubulin gene sequences obtained from A. ochraceus and A. westerdijkiae were exploited by Morello et al. (2007) for designing species-specific primers for A. westerdijkiae detection in coffee beans (Bt2Aw-F/Bt2Aw-R). By using this primer-pair a 347 bp-amplicon was visualized in all A. westerdijkiae isolates but no PCR assay product was observed from A. ochraceus isolates. The Bt2Aw primers were successfully applied for detecting the 347 bp-amplicon when using DNA collected from coffee beans inoculated with A. westerdijkiae.

Although conventional PCR assay is a valuable tool for detection and monitoring mycotoxigenic fungi, it is not appropriate to quantify a given fungus species in a food sample. Small difference in reaction efficiency per cycle can result in a substantial difference in the final product quantity and so the extrapolation to the initial concentration of the template in the sample is very difficult to do (Hill, 1996). Fortunately, the introduction of the Real-Time PCR technology has increased the reliability of PCR results compared to those obtained by conventional methods, thus opening new avenues for quantifying ochratoxigenic fungi in food. Real-Time PCR is more sensitive than classical PCR and does not require gel electrophoresis. The analysis can be concluded with less than 5h. This attribute of Real-Time PCR significantly reduces time and manual labor, making it appropriate for large-scale analyses. The use of fluorophores is common to most of these methods and is described in detail by Boysen et al. (2000). By using Real-Time PCR it is possible to detect an increase in fluorescence emission during the reaction that is proportional to the initial copy number of the target sequence. The initial amount of template DNA is inversely proportional to a parameter measured for each reaction, which is denoted the threshold cycle (C_t). The C_t value is the PCR cycle when the fluorescence signal increases above the background threshold. Application of this method to natural samples provided an estimate of infection by a give species. Morello et al. (2007) evaluated the potential of the Real-Time PCR approach for quantification of this species in coffee beans. Green coffee beans were inoculated with 10⁶ A. westerdijkiae conidia and incubated for 192 h. DNA extraction and colony-forming unit (cfu) assay were performed each 48h. It was observed high correlation between the cfu data and the fungal DNA content in coffee beans. The authors also assessed the sensitivity of this method in order to detect A. westerdijkiae in coffee beans. According to their data positive signal may be obtained with less than 10 copy number of A. westerdijkiae haploid genome. This value also means less than 10 haploid genomes per 0.1 g of coffee beans. Real-Time PCR sensitivity level was more than 100 times higher than the cfu technique. Patiño et al. (2005) developed specific PCR assay for detection of A. ochraceus species based on ITS sequences. The primer-pair denoted OCRA1/OCRA2 was designed on the basis of ITS sequence comparison of several strains of Aspergillus species. The specificity of the primer-pair was tested on a number of Aspergillus, Penicillium, Cladosporium, Botrytis, and Alternaria strains commonly associated with grapes, cereals, and coffee. 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. According to the authors the sensitivity of PCR assay based on ITS sequences was higher (1 and 10 pg of DNA template per reaction) than primers based on single copy gene (0.1 and 1 ng of DNA template per reaction). The authors do not mentioned about the new species A. westerdijkiae. Probably the primer-pair proposed does not distinguish between A. westerdijkiae and A. ochraceus.

The ochratoxigenic species A. carbonarius and A. niger belong to section Nigri, which is an important group of species in food mycology. As commented by Samson et al. (2007), black aspergilli are one of the more complex groups concerning their classification and identification, and numerous taxonomic schemes have been proposed. The differences between some species belonging to section Nigri are very slight and their identifications require molecular analysis.

Specific PCR assay for detection of A. carbonarius species was developed by Patiño et al. (2005) based on ITS sequences. The primer-pair denoted CAR1/CAR2 was able to provide an amplicon of 420 bp only when genomic DNA from A. carbonarius strains was used. Schmidt et al. (2004) used AFLP to detect specific markers for A. carbonarius. A certain number of amplified fragments were found to be specific to this species. The marker fragments were cloned and sequenced and used to design a primer-pair specific to detect this species. The primer-pairs A1B-fw/A1B-rv and C1B-fw/C1B-rv provided amplicons of 189 bp and 351 bp, respectively, in all A. carbonarius isolates tested. Based on alignment of calmodulin (cmdA) gene sequences, Perrone et al. (2004) identified regions suitable for design specific PCR primers for the detection of A. carbonarius strains. The primer-pair (CARBO1/2) produced a PCR product of 371 bp and the sensitivity was about 12 pg when using pure total genomic DNA. Although the PCR assay was useful in screening isolates of black aspergilli from grapes, the authors did not use it to detect A. carbonarius strains directly from grape samples.

Several strains representing closely related black aspergilli, i.e. A. carbonarius, A. niger, A. foetidus, and A. tubingensis were analyzed by RAPD with the aim of developing species-specific primers for the detection of A. carbonarius in coffee beans (Fungaro et al., 2004a). An example of a typical RAPD pattern is given in Figure 1. Some DNA bands were present in all A. carbonarius strains and absent in all strains of A. niger and A. tubingensis. One of these bands was cloned and sequenced, and then used to design a primer-pair specific to A. carbonarius (OPX7₈₀₉-F/OPX7₈₀₉-R). By using this primer-pair the authors successfully detected an amplicon of 809 bp when DNA from coffee beans infected with A. carbonarius strains was used. No cross-reaction was observed using DNA from coffee beans infected with closed related black aspergilli. Similarly, based on RAPD markers, Sartori et al. (2006) developed specific primers for A. niger detection. The primer-pair denoted OPX7_372F/ OPX7_372R provided an amplicon of 372 pb in all A. niger stricto sensu isolates, and no amplification product was observed in reactions using DNA from related species. This PCR assay was successfully applied for detecting A. niger in coffee beans.

Because A. ochraceus (now A. westerdijkiae), A. carbonarius and A. niger are the major species for colonizing Brazilian coffee beans and producing OTA our research group developed a multiplex PCR assay (m-PCR) useful to detect the three target fungi species directly from sample of this commodity (Sartori et al., 2006). The m-PCR is a procedure that allows the simultaneous amplification of more than one target sequence in a single PCR reaction, decreasing the number of reaction to be performed to assess the possible presence of different species in a food sample. The authors firstly analyzed the value of the m-PCR assay with DNA obtained from coffee beans inoculated with these three species. The Figure 2, shows the amplification profiles using simultaneously the primer-pairs designed for A. ochraceus, A. carbonarius, and A. niger. Amplification products of 260 bp, 809 bp and 372 bp, in a single PCR reaction confirmed the presence of A. ochraceus, A. carbonarius, and A. niger, respectively. The usefulness of the m-PCR assay was also analyzed using coffee bean samples collected in farm.

As above mentioned, sequences of functional genes may also be used as target for designing PCR primers to detect mycotoxigenic fungi. However, in contrast to others mycotoxin, the OTA biosynthetic pathway was not well characterized in any of the OTA-producing species and consequently the genes that encode enzymes involved in the biosynthesis of this secondary metabolite are poor known. Based on OTA structure the necessity of various enzymes catalyzing key reactions in the formation of OTA can be expected and some teams of researchers are looking for the genes related to OTA biosynthesis (Lebrihi et al., 2003; Geisen et al., 2004; Atoui et al., 2006; O'Callaghan et al., 2006; Bogs et al., 2006). A polyketide synthase can be postulated as involved in OTA biosynthesis, because isocoumarin group of the OTA is probably a pentaketide formed from acetate and malonate via a polyketide synthesis pathway (O'Callaghan et al., 2003). The diversity of polyketide synthase genes has been investigated (Atoui et al., 2006) in A. carbonarius. Two non conserved sequences in the acyltransferase domain of a polyketide synthase gene, denoted Ac12RL3, were used as a target sequence to detect specifically A. carbonarius by PCR assay. The primer-pair, Ac12RL_OTAF/ Ac12RL_OTAR generated a 141 bp PCR product in all A. carbonarius isolates studied and none of the other species gave a positive result with this PCR primer set (Atoui et al., 2007). This specific primer-pair was successfully employed for the direct quantification of A. carbonarius in grape samples but not in coffee beans. With the same objective, i.e. to quantify A. carbonarius in grape samples, Atoui et al. (2006) used a specific primer-pair (Ac12RL_OTAF/Ac12RL_OTAR) that was designed from the acyltransferase (AT) domain of the polyketide synthase sequence (Ac12RL3) to amplify 141 bp PCR products. By using Real-Time PCR conjugated with SYBR® Green I dye, the authors found a positive correlation (r²=0.81) between A. carbonarius DNA content and OTA concentration in 72 grape samples. Table 1 summarizes the primer-pairs sequences designed for detection OTA-producing fungi in coffee beans.

Several analytical methods for the detection of OTA exist and the level of this mycotoxin can readily be measured very accurately in food, but this kind of analysis only returns a positive result once the toxins have been formed. Similarly several methods for the detection of ochratoxigenic species have been described, but the presence of an ochratoxigenic fungal species in a sample does not ultimately mean that the toxin itself is present. As occurs with other mycotoxins, the substrate on which the fungus grows, as well as the moisture content and temperature can determine if the mycotoxin will be produced. Moreover, there are great differences between strains of an ochratoxigenic species concerning their ability for toxin production. We have previously shown that 30% of A. westerdijkiae strains isolated from coffee beans are unable to produce detectable amounts of OTA (Morello et al., 2007). Based on these points, more important for a meaningful monitoring of OTA in food would be the measurement of the rate of expression of mycotoxin genes, because these genes are frequently expressed some days prior to the mycotoxins production and thus would allow an early warning (Schmidt-Heydt and Geisen, 2007).

The first relevant report of the cloning and characterization of putative polyketide synthase gene (pks) from Aspergillus involved in OTA biosynthesis was done by O'Callaghan et al. (2003). The authors used a molecular strategy denoted suppression subtractive hybridization PCR-based and discovered that this pks gene is expressed only under OTA permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted was not able to synthesize OTA. Afterward the authors examined OTA production by A. ochraceus grown under different nutritional and environmental conditions. Quantifications of transcript accumulation of the pks gene showed that pks transcription is tightly linked to OTA production (O'Callaghan et al., 2006). However it is important to state that although this gene is really more expressed by a positive strain under OTA permissive conditions, no information was available about the expression of this gene by OTA-non-producing strains.

Preliminary investigation carried out by our group showed that the pks gene, described by O'Callaghan et al. (2003), is in fact significantly more expressed by A. westerdijkiae when grown in permissive conditions to the production of OTA than when grown in restrictive conditions to the production of this toxin. However, when a negative strain is cultivated in permissive conditions to the production of OTA, the pks gene is similarly express as the positive strain; though there is no OTA production (not published data). This fact is probably because other secondary metabolites can share this pks gene. This observation means that elucidation of differentially expressed genes between OTA-producing strains and OTA-non-producing strains is still required.

Representational Difference Analysis (RDA), as firstly described by Lisitsyn et al. (1993) is a powerful differential hybridization method to identify unique DNA sequences out of two complex and highly related genomes. With equal success, this approach was adapted by Hubank and Schatz, (1994) aiming at the identification of specific genes differentially expressed (cDNA RDA), and it has been employed for expression profiling in fungi species. We are using RDA approach in order to detect differences in gene expression between A. westerdijkiae

OTA-producing and non-producing strains grown under permissive conditions for OTA production. Until now, we identified two genes encoding oxidoreductases (P450-AL and OXI-1) that are markedly differentially expressed between OTA producing and non-producing strains of A. westerdijkiae (unpublished data). Comparative proteomics analysis are also in progress in our laboratory in order to detect differences in protein expression between A. westerdijkiae OTA-producing and non-producing strains grown under permissive conditions for OTA production. Some successful was already obtained as illustrated in Figure 3.

Finally, our group has been engaged in an effort to find genome differences that could be useful for distinguishing toxigenic from non-toxigenic A. niger strains. Very recently we described that one pks gene is specific to OTA-producer strains. As this species is frequently applied for several industrial purposes and it is often found in a number of agricultural products, we are supposing that this pks gene will be very useful as a molecular marker for discriminating A. niger strains concerning their ability to produce OTA (unpublished data). This approach will guarantee that products of fungal fermentation or maturation are ochratoxin-free.

Concluding, over the last 5 years, several molecular assays for identification and fast detection of ochratoxigenic species without the need of isolating pure cultures were published. These assays include conventional PCR, multiplex PCR, Real Time PCR and RT-Real Time PCR.

We are optimistic that in the near future the molecular technologies will be useful as a preventive approach to minimize the entry of ochratoxin into the food chain.

ACKNOWLEDGMENTS

Our study was supported (grants and fellowships) by the following Brazilian institutions: Fundação Araucária, Conselho Nacional de Pesquisa (CNPq), Financiadora de Estudos e Projetos (FINEP), Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES), Rede Integrada de Biotecnologia Aplicada ao Desenvolvimento Sustentável da Agricultura Familiar no contexto do Agro-negócio Paranaense (BIOAGROPAR) and Laboratório de Apoio à Pesquisa Agropecuária (LAPA).

RESUMO

Dentre as micotoxinas que contaminam produtos destinados à alimentação humana e animal, a ocratoxina A (OTA) é uma das mais freqüentemente encontrada. A sua ocorrência em vários produtos agrícolas tem sido considerada um problema de amplitude mundial. Esta toxina é produzida principalmente por fungos do gênero Aspergillus. A OTA tem efeitos nefrotóxico, imunossupressor e carcinogênico. A contaminação de alimentos com esta toxina representa risco para a saúde animal e humana. Nos últimos cinco anos, vários investigadores têm desenvolvido métodos moleculares para identificação e quantificação de fungos produtores de OTA em amostras de grãos de café. O objetivo desta revisão é apresentar e discutir as várias estratégias desenvolvidas recentemente para a detecção de fungos potencialmente produtores de OTA.

REFERENCES

Atoui, A., Dao, P., Mathieu, F., Lebrihi, A. (2006), Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A. Mol Nutr Food Res., 50, 448-493.

Atoui, A., Mathieu, F., Lebrihi, A. (2007), Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR. Int J Food Microbiol., 115, 313-318.

Bennett, J. W., Klich, M. (2003), Mycotoxins. Clin Microbiol Rev., 16, 497-516.

Battilani, P., Pietri, A., Bertuzzi, T., Languisco, L., Giorni, P., Kozakiewicz, Z. (2003), Occurrence of ochratoxin A - producing fungi in grapes grown in Italy. J Food Prot., 66, 633-636.

Bayman, P., Baker, J. L., Doster, M. A., Michailides, T. J., Mahoney, N. E. (2002), Ochratoxin production by the species Aspergillus ochraceus group and Aspergillus alliaceus. Appl Environ Microbiol., 68, 2326–2329.

Bogs, C., Battilani, P., Geisen, R. (2006), Development of a molecular detection and differentiation system for ochratoxin A producing Penicillium species and its application to analyse the occurrence of Penicillium nordicum in cured meats. Int J Food Microbiol., 107, 39-47.

Boysen, M. E., Jacobsen, J., Schnurer, J. (2000), Molecular identification of species from the Penicillium roqueforti group associated with spoiled. Appl Environ Microbiol., 66, 1523-1526.

Comission Regulation No. 1881/2006 Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L 364/4-364/24. CX/CF 08/2/1, 19 December 2006.

Frisvad, J. C., Frank, J. M., Houbraken, J., Kujipers, A. F. A. (2004), New ochratoxin A producing species of Aspergillus section Circumdati. Stud Mycol., 50, 23-43.

Fujii, S., Ono, E.Y.S., Ribeiro, R.M.R., Assunção, F.G.A., Takabayashi, C.R., Oliveira, T.C.R.M., Itano, E.N., Ueno, Y., Kawamura, O., Hirooka, E.Y. (2007), A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, toasted and instant coffee. Braz. Arch. Biol. Tech., 50, 349-359.

Fungaro, M. H. P., Vissotto, P. C., Sartori, D., Vilas-Boas, L. A., Furlaneto, M. C., Taniwaki, M. H. (2004a), A molecular method for detection of Aspergillus carbonarius in coffee beans. Curr Microbiol., 49, 123-127.

Fungaro, M. H. P., Magnani, M., Vilas-Boas, L. A., Vissotto, P. C., Furlaneto, M. C., Vieira, M. L. C., Taniwaki, M. H. (2004b), Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences. Can J Microbiol., 50, 985-988.

Geisen, R. (1998), PCR methods for the detection of mycotoxin producing fungi. In: Bridge, P. D., Arora, K. K., Reddy, C. A., Elander, R. P. (eds) Applications of PCR in micology. Cambridge, UK: CAB International, 243-266.

Geisen, R., Mayer, Z., Karolewiez, A., Farber, P. (2004), Development of a real time PCR system for detection of Penicillium nordicum and for monitoring ochratoxin A production in foods by targeting the ochratoxin polyketide synthase gene. Syst Appl Microbiol., 27, 501-507.

Hill, W.E. (1996), The polymerase chain reaction: applications for the detection of foodborne pathogens. Crit Rev Food Sci Nutr., 36, 123-173.

Hubank, M., Schatz, D. G. (1994), Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res., 22, 5640-5648.

Jorgensen, K. (1998), Survey pork, poultry, coffee, beer and pulses for ochratoxin A. Food Addit Contam., 15, 550-555.

Jorgensen, K., Jacobsen, J. S. (2002), Occurrence of ochratoxin A in Danish wheat and rye, 1992-1999. Food Addit Contam., 19, 1184-1189.

Lebrihi, A., Mathieu, F., Borgida, L. P., Guyonvarch, A. M. (2003), Method for the detection of ochratoxin A or citrinin-producing fungi. European patent nº. EP1329521.

Lisitsyn, N. A., Lisitsyn, N. M., Wigler, M. (1993), Cloning the differences between two complex genomes. Science., 259, 946-951.

Morello, L. G., Sartori, D., Martinez, A. L. O., Vieira, M. L. C., Taniwaki, M. H., Fungaro, M. H. P. (2007), Detection and quantification of Aspergillus westerdijkiae in coffee beans based on selective amplification of β-tubulin gene by using real-time PCR. Int J Food Microbiol., 119, 270-276.

O'Callaghan, J., Caddick, M. X., Dobson, A. D. (2003), A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus. Microbiology., 149, 3485-3491.

O'Callaghan, J., Stapleton, P. C., Dobson, A. D. (2006), Ochratoxin A biosynthetic genes in Aspergillus ochraceus are differentially regulated by pH and nutritional stimuli. Fungal Genet Biol., 43, 213-221.

Patiño, B., González-Salgado, A., González-Jaén, M. T., Vázquez, C. (2005), PCR detection assays for the ochratoxin producing Aspergillus carbonarius and Aspergillus ochraceus species. Int J Food Microbiol., 104, 207-214.

Perrone, G., Susca, A., Stea, G., Mulé, G. (2004), PCR assay for identification of Aspergillus carbonarius and Aspergillus japonicus. Eur J Plant Pathol., 110, 641–649.

Petzinger, E., Weindenbach, A. (2002), Mycotoxins in the food chain: the role of ochratoxins. Livest Prod Sci., 76, 245-250.

Russell, R., Paterson, M. (2006), Identification and quantification of mycotoxigenic fungi by PCR. Process Biochem., 41, 1467-1474.

Samson, R. A., Noomin, P., Meijer, M., Houbraken, J., Frisvad, J.C., Varga, J. (2007), Diagnostic tools to identify black aspergilli. Stud Mycol., 59, 129-145.

Sartori, D., Furlaneto, M. C., Martins, M. K., Paula, M. R. F., Pizzirani-Kleiner, A., Taniwaki, M. H., Fungaro, M. H. P. (2006), PCR method for the detection of potential ochratoxin-producing Aspergillus species in coffee beans. Res Microbiol., 157, 350-354.

Schmidt, H., Ehrmann, M., Vogel, R. F., Taniwaki, M. H., Niessen, L. (2003), Molecular Typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP. Syst Appl Microbiol., 26, 138-146.

Schmidt, H., Taniwaki, M. H., Vogel, R. F., Niessen, L. (2004), Utilizationof AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples. J Appl Microbiol., 97, 899-909.

Schmidt-Heydt, M., Geisen, R. (2007), A microarray for monitoring the production of mycotoxins in foods. Int J Food Microbiol., 117, 131–140.

Smith, J. E., Henderson, R. S. (1991), Mycotoxins and animal foods. London: CRC Press, 816-841.

Taniwaki, M. H., Pitt, J. I., Teixeira, A. A., Iamanaka, B. T. (2003), The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. Int J Food Microbiol., 82, 173-179.

Taniwaki, M. H., Teixeira, A. A., Iamanaka, B. T., Fungaro, M. H. P., Palácios-Cabrera, H. A., Pitt, J. I. (2008), An Update on Ochratoxin A in Coffee After 10 years of Research Paper presented at 22nd International Conference on Coffee Science, Campinas, São Paulo, Brazil.

Visconti, A., Pascale, M., Cewntonze, G. (2001), Determination of ochratoxin A in wine and beer by immunoaffinity columm cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study. J AOAC Inter., 8, 1818-1827.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van De Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M. (1995), AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res., 23, 4407-4414.

Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., Tingey, S. V. (1990), DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18, 6531-6536.

Publication Dates

• Publication in this collection
  08 Feb 2010
• Date of issue
  Nov 2009