Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition.
Project description:Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production. A total of 40 potentially mycotoxigenic fungi isolated from different food commodities, as well as the genes that are involved in the mycotoxin synthetic pathways, were analyzed. For fungal identification, oligonucleotide probes were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotides directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. The oligonucleotides selected for fungal identification and the oligonucleotides specific for toxin producing genes were spotted onto microarray slides. The diagnostic microarray developed can be used to identify potentially mycotoxigenic fungi as well as genes leading to toxin production in both laboratory and food samples offering an interesting potential for microbiological laboratories. Keywords: Development of a diagnostic microarray for the identification of potentially mycotoxigenic fungi as well as genes leading to toxin production, 40 food-borne fungi, mycotoxins Development of a diagnostic array for the identification of food-borne fungi and their potential mycotoxin-producing genes. Oligonucleotide probes to be printed onto the array were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotides directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. Analysis was performed with 40 fungal cultures were obtained from the Agricultural Research Council culture collection (ARC), Pretoria, South Africa.an in-house spotted oligonucleotide microarray. The identity of each fungus was confirmed by standard laboratory procedures. For DNA isolation, the fungal strains were grown on 1.5% malt extract agar at 25°C for 1-2 weeks and total genomic fungal DNA was extracted following the DNA extraction protocol described by Raeder and Broda (1985). The internal transcribed spacer oligonucleotides ITS1, ITS3 and ITS4 were used as a reference for normalization of all spot intensity data.Samples were fluorescently labelled with Cy5 dye by using a Cyâ¢Dye Post-labelling Reactive Dye Pack and wre hybridized to the oligonucleotide microarray overnight. Two biological and one technical replicate (using independent labelling reactions) was performed, each replication consisting of a reverse labelling experiment.