The microarray technique is thus analogous to performing many PCR reactions and hybridization reactions at the same time and has the advantage of being versatile . The aim of this study was to develop a diagnostic microarray for the identification of single strains of food-borne fungi that are most prevalent in South African #see more randurls[1|1|,|CHEM1|]# food commodities, and to detect the ability of these fungi to produce
mycotoxins in laboratory and food samples. A total of 40 food-borne fungi isolated from different foods that belong to the genera Alternaria, Aspergillus, Bipolaris, Claviceps, Curvularia, Diplodia, Drechslera, Eurotium, Fusarium, Penicillium and Pithomyces, were used. For fungal discrimination, the polymorphisms of the internal transcribed spacer (ITS) regions and the elongation factor 1- alpha (EF-1 α) gene were exploited for the design of the oligonucleotide probes. The specificity of a probe was increased in some instances by substituting an oligonucleotide with a high affinity DNA analogue known as locked nucleic acid (LNA). A locked nucleic acid nucleotide analogue consists of a 2′-O,4′-C methylene bridge and locks the LNA structure into a rigid bicyclic formation and displays unprecedented hybridization affinity towards complementary DNA and RNA . It is most disruptive, and thus gives a better signal, in a centre position. For the detection
of fungi that can produce mycotoxins, oligonucleotide probes for the genes leading to mycotoxin production were selected
from public databases and included in the oligonucleotide array. The combination of ITS, EF-1 PF-02341066 cell line α and mycotoxin genes on the same array was evaluated for the potential of the array to identify the forty fungal isolates and the genes involved in pathways leading to toxin production. Results Probe design A 96-probe oligonucleotide microarray was constructed for the simultaneous Sodium butyrate detection and identification of potentially mycotoxigenic fungi. Probes for the array were designed by exploiting the polymorphisms of the internal transcribed spacer (ITS) regions of the rRNA complex. Amplification of fungal DNA with the universal fungal primers ITS1 and ITS4 and subsequent sequence analysis allowed the differentiation of most of the fungal species studied. Several unique polymorphisms (sequence data can be found in GenBank with accession numbers [GenBank:FJ864706, GenBank:FJ864709, GenBank:FJ864710, GenBank:FJ864708, GenBank:FJ864711, GenBank:FJ864703, GenBank:FJ864704, GenBank:FJ864705, GenBank:FJ864707, and GenBank:FJ864712]) could be identified within the PCR products generated for each fungal species. However, amplification of the Fusarium species showed no significant differences between the sequences of the PCR products generated with the ITS primers. Therefore, the elongation factor 1-alpha (EF-1 α) gene was used for the identification of polymorphisms in Fusarium species and for the design of unique species- or genus-specific probes.