Belén Patiño

PCR detection assay of fumonisin-producing Fusarium verticillioides strains.

Fusarium verticillioides is considered to be the main source of fumonisins, a group of toxins that contaminate commodities and result in chronic and acute diseases affecting humans and animals. The detection and control of this species is crucial to prevent fumonisins from entering the food chain. The objective of the present research was to develop a specific, sensitive, and robust PCR assay to detect F. verticillioides strains using two pairs of specific primers for F. verticillioides, which have been designed on the basis of the intergenic spacer region of the rDNA units. The first pair of primers was F. verticillioides species specific, whereas the second pair of primers detected fumonisin-producing F. verticillioides strains. This second pair of primers allowed for the discrimination between the major group of F. verticillioides strains, fumonisin-producing strains that are mainly associated with crops, and a minor group of strains, non-fumonisin-producing strains that are associated with bananas. Fifty-four strains of F. verticillioides from different geographical regions and hosts were tested using both sets of primers. Sixteen additional Fusarium species were examined. The specificity of the primer sequences provides the basis for a simple, rapid, accurate, and sensitive detection and identification method of this fungal species that represents a risk for human and animal health.

Genetic markers for the analysis of variability and for production of specific diagnostic sequences in fumonisin-producing strains of Fusarium verticillioides

Fusarium verticillioides(Gibberella moniliformis, Gibberella fujikuroi mating population A) is the main source of fumonisins, a group of toxins which contaminates commodities, causing chronic and acute diseases in humans and animals. Fumonisins are produced during colonisation and infection of host plants even when disease symptoms are not recognisable. Early detection and control of F. verticillioides is crucial to prevent fumonisins from entering the food chain. DNA-based strategies have been used to search for markers to develop sensitive, robust and specific diagnostic assays, mainly based on PCR. The different approaches used, based either on DNA markers unrelated to fumonisin production or on information about the genes involved in fumonisin production, are described and discussed. The ability of these methods to discriminate between the two populations occurring within F. verticillioides, fumonisin-producing and fumonisin non-producing strains, is also addressed.

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR.

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10(6)spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.

ITS-based detection and quantification of Aspergillus ochraceus and Aspergillus westerdijkiae in grapes and green coffee beans by real-time quantitative PCR.

Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (OTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point of the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production.

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 102 spores after 16 h of incubation.

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp.

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.

Specific detection of Aspergillus carbonarius by SYBR® Green and TaqMan® quantitative PCR assays based on the multicopy ITS2 region of the rRNA gene.

Agroproducts contaminated by ochratoxin A (OTA) represent a risk for human and animal health and, therefore, maximum limits have been established by Food Safety Authorities. Reduction of OTA contamination may be accomplished by early detection of OTA-producing fungal species using rapid, specific and sensitive detection and quantification by PCR-based methods. Aspergillus carbonarius is one of the most important OTA-producing species, in particular in grapes and derivatives from Mediterranean regions. In this work, highly efficient quantitative PCR assays using SYBR Green I and TaqMan methods were developed for specific detection of A. carbonarius to be used in grapes. The primers and the TaqMan probe were based on the internal transcribed region 2 multicopy region (internal 2 sequence of the rRNA gene). The specificity and sensitivity of both assays were tested on genomic DNA mixtures of several A. carbonarius strains and other fungal species frequently present in grapes. Both methods were also compared using grapes inoculated with different spore concentrations of A. carbonarius, detecting up to 0.4 pg DNA g(-1) grape berries. The efficiency and sensitivity of both methods were comparable and only the lower cost of SYBR Green might favour its use in routine screenings.

Discrimination of the main Ochratoxin A-producing species in Aspergillus section Circumdati by specific PCR assays.

Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. Some of the main OTA-producing species belong to Aspergillus section Circumdati, whose taxonomy has been lately revised with the description of new species. The high morphological similarity of these species (Aspergillus ochraceus, Aspergillus steynii and Aspergillus westerdijkiae) makes difficult discrimination among them and with respect to the other species included in the same section unable to produce OTA. In this work, PCR assays specific for the OTA-producing species, A. ochraceus, A. steynii and A. westerdijkiae, were developed on the basis of the multicopy ITS regions of the rDNA. The assays were tested in a wide sample of isolates from diverse origins and culture collections, confirming their specificity, and revealing that the current identification of strains from section Circumdati might need a revision.

Fumonisin production by Gibberella fujikuroi strains from Pinus species

Fumonisins are important mycotoxins basically produced by strains from the Gibberella fujikuroi species complex (with anamorphs in Fusarium genus) which contaminate food and feed products representing a risk to human and animal health. In this work, we report for the first time the fumonisin production of Fusarium moniliforme Sheldon strains associated to edible pine nuts of Pinus pinea. P. pinea is an important and widely distributed Pinus species in the Mediterranean area where their pine nuts are consumed raw or slightly processed in diverse food products. In this work, characterization and further identification of those strains were performed by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the intergenic spacer region of the rDNA (IGS) with the aid of the eight mating populations (A-H) described for G. fujikuroi species complex. The method was powerful to detect polymorphism, allowing discrimination between individuals and could be used to study the genetic relationships among them and within the G. fujikuroi species complex. Fusarium strains associated to Pinus radiata were also included in the present study. These strains did not produce fumonisins and showed no close relation with the strains isolated from P. pinea. The approach used in this work was rapid and proved to be efficient to assist identification and to characterize and analyse relatedness of new isolates within the G. fujikuroi species complex.

Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.