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Risk assessment and integrated ochratoxin a (ota) management in grape and wine

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The incredible explosion of knowledge production in Biology during the two last decades has created a critical need for bioinformatics instruments able to manage data and facilitate their retrieval and analysis. Hundreds of biological databases have been produced and integration of biological data from these different resources is extremely necessary when we want to focus our efforts towards the study of a particular layer of biological knowledge. Molecular sequences and biological data on gene and their functional products are publicly available from a wide variety of databases. A part from primary and genomic databases (Embl, Swissprot, Ensembl, etc.), in which these kind of information are dispersed among all the others, many specialized databases have been developed collecting data from external heterogeneous data resources, including literature references, and in some cases also adding information coming from in-silico and/or experimental studies of gene and/or protein functional characterization. As far as those databases dedicated to sequences, no information are available on databases dedicated to AFLP automated data, in collaboration with a bioinformatics society, we have developed a specific database named “AFLP Manager” able to recognize and download directly the ABI files generated from the GeneScan Collection software (PE Applied Biosystems) after the separation on ABI Prism 310 automated DNA sequencer. The AFLP Manager after loading the ABI prism data is able to recognize each information on the sample (peaks, time of run, areas of peaks, etc.). This database was developed in a DBMS free in MSQL and it is able to collect, modify, compare data, make query and export a final data set in a binary matrix useful for statistical analyses. Actually the database is available on the web at the following URL:http://server.ispa.cnr.it/ITEM/AflpManager.
The highly conserved regions of calmodulin gene were considered as target sequences for the assay. In an alignment of sequences representing Aspergillus spp. occurring on grapes, primers and probes sequences were chosen. The primers and probe sequences were completely conserved among all sequences examined within the A. carbonarius strains. Primer/probe combination successfully amplified DNA extracted from pure cultures of A. carbonarius fungal species, whereas no fluorescence could be measured with any of the other species within the Aspergillus genera. Occasionally, the fluorescence value of negative control (containing water instead template) started to increase after 36 - 38 cycles. This is not unusual when use SYBR Green and is caused by primer-dimers formation (especially at low level of target template). Even though this effect was almost eradicated by reducing the primer concentration, any increase in fluorescence after this point was deemed unreliable and not used to detect or quantify samples. It was avoided using a sequence specific assay, as TaqMan method. The TaqMan real time PCR is based on the cleavage of an internal probe by the 5’-/3’ endonuclease activity of the Taq polymerase. During each cycle of the extension phase one molecule of the reporter dye is released for each molecule amplified resulting in generation of fluorescent emission of the reporter dye. The threshold cycle (Ct values) indicates the increase of reporter fluorescence and it is the number of cycles before the fluorescence emitted passes a fixed limit. The log10 of the number of targets initially present is proportional to the Ct value and can be measured using a standard curve. To develop a standard curve for the quantitative PCR reactions were used serial dilutions of A. carbonarius strains. A pure culture of A. carbonarius was used to extract the DNA and make two fold dilutions for obtaining the standard curve useful for Real Time PCR. Using the TaqMan method, large numbers of samples can be processed allowing a much more intensive monitoring of the population dynamics. To evaluate intra-assay variability, each dilution was assayed in triplicate. Relative standard deviations of the Ct values within the same sample was lower than 1.5%. Application of this method to a known conidial suspension (CFU/ml) provided a positive correlation between CFU and haploid genome weight. Genomic DNA weight of A. carbonarius (not known) was assumed to be similar to that of A. niger (4.13 x 10-5ng, for a haploid genome) reported in the website http://www.broad.mit.edu/annotation/fungi/fgi/history.h tml. To verify this assumption an absolute quantification of total DNA was made for DNA extracted from a known conidial suspension (3x102 CFU/ul) of A. carbonarius, by real-time quantitative PCR assay. For a suspension of 3x105 conidia 10.95ng of total DNA was obtained, corresponding to 3.65x10-5 ng for a single CFU or conidium, which is very close to the genome weight of A. niger (35.9 Mb). In order to study the usefulness of the TaqMan PCR assay as a tool in food quality/safety control the quantification of A. carbonarius DNA was carried out in triplicate on 15 samples of grapes having a known content of OTA. The regression curve of A. carbonarius DNA content versus OTA content showed a very good correlation (R2=0.92), despite the finding of low levels of DNA in 4 ochratoxin-free samples.
The visual inspection of the aligned calmodulin partial gene sequences readily identified unique regions within the amplified fragment, (e.g. in the range of 350-420 base pairs) that has been exploited in the development of generic PCR-based assay for the detection of A. carbonarius and A. japonicus/aculeatus. Using the genetic sequence variation founded in this region, all primers were designed to operate at high annealing temperatures (58°C), thereby preventing the co-amplification of non specific target of DNA. In calmodulin sequences, we observed 99,98 % identity for strains of A. carbonarius, and 99,40 % identity for strains of A. japonicus and A. aculeatus analysed. Specific primer pairs amplified in A. carbonarius a fragment of 371 bp length and in A. japonicus a fragment of 583 bp length. To determine the diagnostic value of the primer set in a large scale, experiments were started to test its usability on a large number of isolates: 30 strains each of the target species, 42 strains of other closely related species within the black aspergilli and others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species. Aspergillus japonicus strains were not amplified by A. carbonarius-specific PCR primers (CARBO1/2), and A. carbonarius strains were not amplified by A. japonicus -specific PCR primers (JAPO1/2). None of the others Aspergillus section Nigri were amplified by the two sets of primers with the exception of the four strains of A. aculeatus which were amplified by the specific primers JAPO1/2. No product was amplified from any fungal/yeast tested. The primers were also tested against others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species, without any amplifications
The data of AFLP analysis performed on 300 strains with the primer A showed a clear pattern’s peaks for the three species main occurring on grapes. So we are able to construct a dendrogram of similarity that evidenced the grouping in three distinct clusters of the strains belonging to A. carbonarius, A. niger group and A. japonicus/A. aculeatus . These result were successively confirmed by the AFLP analyses made with other three primers (B, C and D), all the results obtained gave good fingerprints useful to construct dendrograms that clearly separate the three main groups of the Black Aspergilli (Fig. 5). The AFLP banding pattern was variable from 20-25 bands for A. carbonarius and A. japonicus aggregate to 30-40 bands for A. niger aggregate, confirming the higher genetic variability among this species aggregate as was showed by sequence analyses. On the basis of these great variability found in the A. niger aggregate strains the AFLP analysis revealed the presence of two main clusters within this species aggregate that seem clearly separated, suggesting the possible split in two different species of this two groups of A. niger strains.

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