The development of novel aptamers for the detection of T-2 and HT-2 mycotoxins

Abstract

Contamination of food and feed with fungal secondary metabolites, such as the mycotoxins T-2 and HT-2 produced by Fusarium spp., poses a significant risk to human and animal health. Rapid and portable detection methods based on the use of biomolecules such as aptamers are highly desirable to ensure food safety. The aim of this study was to develop novel DNA aptamers with specificity for the low molecular weight mycotoxins, T-2 and HT-2. A combination of three experimental approaches were evaluated to achieve this aim. First, selection of aptamers through a modified Capture SELEX protocol using lambda exonuclease for digestion of dsDNA to ssDNA following PCR amplification. Second, application of high throughput sequencing (HTS) for the characterisation of aptamer candidates. Third, assessment of aptamer binding through colorimetric gold nanoparticle (AuNPs) aggregation assays. Improved ssDNA recovery (>55%) suitable for use in subsequent selection rounds significantly reduced Capture SELEX process time. HTS analysis within the T-2 and HT-2 selections successfully enabled identification of unique sequences as aptamer candidates. Six novel aptamers (three from the T-2 and three from the HT-2 selection rounds), originating from different selection rounds, were obtained. Two of these aptamers were cross-selected between the targets. Six aptamers (Apta20-01, Apta20-02, Apta20-04, Apta20-57, Apta20-90 and Apta20-100) were found to specifically bind to both targets over a range of concentrations T-2 (0.2µM to 214.3µM) and HT-2 (0.2µM to 235.5 µM), with Apta20-01, Apta20-57 and Apta20-100 showing best performance. Apta20-90 selected from Capture SELEX selection round 1 also demonstrated good performance. Although minimal cross reactivity with non-target mycotoxins (aflatoxin B1 and DON) was observed, the assay proved unsuitable for detection of T-2 and HT-2 in extract derived from one oat sample. Overall, aptamers with target specificity were obtained significantly more rapidly than previously reported Capture SELEX protocols. The protocol has the potential to be applied successfully to other small molecule targets.