Comparison of oligonucleotide arrays and SPR-biacore sensors for fungal detection: tests of Aspergillus carbonarius.

During the time frame of COST853, we presented results on DNA arrays for detection of Fusarium species belonging to the Liseola Section. In this work, we focused the attention on the detection on Aspergillus carbonarius, a common contaminant of several food commodities and the main responsible for ochratoxin A (OTA) accumulation in grapes and wine. OTA is a mycotoxin highly toxic for humans. In order to rapidly identify the A. carbonarius species, specific sequences were selected in the ITS1 and ITS2 region of various Aspergillus species. Two generic primers (P1For, P2Rev) were designed in order to amplify the fungal rDNA (including ITS1 and ITS 2 region). In the DNA macroarray experiments, oligonucleotides specific for A. carbonarius, A. japonicus and A. aculeatus, were provided with an NH2-terminal modification for covalent binding to epoxy-slides (Nexterion, Schott). These immobilised sequences were used as probe to test their specificity in hybridization experiments with the cyanine 3-labeled amplified products. Surface plasmon resonance (SPR) is an emerging technology that enables the label-free detection of biomolecular interactions. In the SPR sensor experiments, the probes were prepared biotinylated in their 5' end and immobilized on a dextran-streptavidin pre-coated gold chip by means of the well known biotin-streptavidin interaction. The probes were tested for biospecific interactions with label-free amplified products. Conclusions: we tested two different methods to verify the specificity of various probes. Fungal DNA amplified with P1For and P2Rev generic primers was used as target. The advantage of the DNA macroarray method consists in the possibility to immobilize a high number of different probes on the same chip, on the contrary, only two probes can be immobilized on the chip in the SPR method. Nevertheless the SPR method has the advantage that the amplified target DNA is label-free. The experiments allowed the identification of the working conditions useful for obtaining specific hybridization signals. The obtained results show that the two methods are both specific and sensitive.