Selection of highly specific aptamers to detect Fusarium graminearum and Fusarium pseudograminearum

The aim of this project was to select highly sensitive and specific aptamer capable of detecting Fusarium graminearum (Fg) and F. pseudograminearum (Fp) species. The aptamer-based testing allows early detection of fungal infection in the field. Fungi are known to change their activity and may often undergo genetic changes. The classic "gene for gene" theory explains the relationship between host plant cultivars and fungal pathogenic races. To avoid the induction of resistance in plant cultivars, fungi changes function of some virulence genes, thus giving rise to new pathogenic strains. Loss of pathogenicity during culture storage is frequently observed in fungi. This could be due to spontaneous mutations or segregation of heterokaryotic or extrachromosomal elements. For these reasons, we isolated fusarium strains from field grown wheat plants that exhibited fusarium-specific symptoms. The stem sections from infected plants were sub-cultured on potato dextrose agar and pure cultures were obtained by serial dilution and spread plating. Subsequently, molecular identification of fusarium species was performed using species-specific primers. Further, to confirm the species of the fungus and the purity of the culture, ITS6 region was sequenced. The BLAST results of sequencing data confirmed isolation of pure cultures of Fg and Fp from filed samples.<br><br> Biosensors are attractive for field testing because they are cost-effective, portable and can be used even by a lay person. Sensors used currently in the agriculture pathogen detection market are based on antibodies, which have low stability and their production is difficult as it involves use of animals. An alternative for antibodies are DNA/RNA aptamers that can be chemically synthesized at a low cost and are thermally stable. Aptamers bind to their target molecules with high affinity and specificity through complementary spatial configuration. Aptamers can bind to proteins, enzymes, biotoxins, or even whole cells. We utilised the repetitive selection-amplification process known as systematic evolution of ligands by exponential enrichment (SELEX) for selection of aptamers specific to Fp and Fg spores. The SELEX process required no prior knowledge of cell surface molecules on the target cell and allows detection of whole live cell.<br><br> The cell SELEX was performed using a commercially available N40 aptamer library with known forward and reverse flanking sequences on the sides of random single-stranded 40 bp DNA sequences (Trilink DNA technologies). Fluorescence Activated Cell Sorting was used to select aptamers binding to fungal spores. The bound aptamers were amplified thrice. Negative selection was used to eliminate those aptamers that might bind to other fungal species infecting wheat. After 3 cycles of positive selection, alternating cycles of negative selection was performed for a total of 9 cycles. Subsequently, the pool obtained was cloned into pCR XL-2-TOPO vector, transformed to E. coli and sequenced. On analysing the sequencing data, 43 and 31 different aptamers binding to Fg and Fp, respectively were identified. These aptamers were analysed using in silico and phylogenetic analysis. Four aptamers with the maximum number of copies were selected from both pools. Out of these, two had high affinity towards both the fusarium species (also present in maximum copies) and two were specific to each of the species. After identifying these four optimal aptamers, these were commercially synthesized and the specificity was confirmed using confocal microscopy and scanning electron microscopy (SEM). In addition, the dissociation constants (Kd) of these four aptamers was determined to select the best one for generating a nano-aptasensor.