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Third generation sequencing and EDNA for detection of aflatoxin production in the soil.

Andres Espindola: Oklahoma State University

<div><em>Aspergillus flavus </em>is a major food safety concern for the food industry. Toxic strains produce varying amounts of the carcinogen aflatoxin B1. This study aims to improve the time-consuming soil sampling, isolation, culturing, toxin detection and exploration of the soil microbiome needed to manage aflatoxin contamination. Third Generation Sequencing (TGS) using a MinION<sup>TM</sup> portable sequencer allows sequencing rapidly metatranscriptomes. Yet, traditional microbiome analyses of TGS samples offers only a hint of the functional characteristics of the microbiome in the soil. E-probe Diagnostic for Nucleic acid Analysis (EDNA) is a bioinformatic pipeline that can infer the production of aflatoxin and detect <em>A. flavus </em>strains rapidly. In this study, the EDNA pipeline was used to measure the presence of atoxigenic and toxigenic <em>A. flavus </em>in soil and the production of aflatoxin. Soil inoculated with AF70 (toxigenic strain) at different titers and AF36 (atoxigenic, control) were incubated at 28 °C. RNA was extracted from the inoculated soil and mRNA sequenced using the MinION<sup>TM</sup>. The sequencing output was analyzed using EDNA transcriptomics and differences were found between titers and aflatoxin gene regulation using e-probes. The obtained results suggest that EDNA can discriminate semi-quantitatively between strain taxonomic units and relative aflatoxin production in soil samples. This is the first time aflatoxin has been directly assessed in situ in the soil environment.</div>