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Accurate detection of three waterborne plant virus genera allows fine screening to species level by High Resolution Melting analysis

Francisco Ochoa Corona: Oklahoma State University

<div>Plant viruses are released into the soil and water from infected and decaying plants, and living root tissues. Stable viruses i.e. <em>Potexvirus</em>, <em>Tobamovirus</em>, and <em>Tombusvirus</em> are reported to contaminate soil and water in forest ecosystems, agricultural fields, hydroponic cultures, and irrigation water. These three virus genera were selected as model groups for their morphological and taxonomical differences. High Resolution Melting (HRM) analysis of RT-qPCR products enabled sensitive detection and discrimination of species with no need for electrophoretic resolution of products. Four assays were developed. The first consisted of three genus-specific sets of primers for single and/or multiplex endpoint RT-PCR for genus detection in water samples. Genus confirmation and screening of virus species was aided by three other sets of genus-specific primers designed for HRM discrimination. Analysis of expected PCR-product sequences using uMELT<sup>SM</sup> allowed prediction of HRM scenarios. Normalized data derived from raw plots of loss of fluorescence vs. temperature and difference graphs showed an accurate discrimination of 11 <em>Potexvirus, </em>11 <em>Tobamovirus</em> and six <em>Tombusvirus</em> with high confidence (<em>p-</em>value 0.003-0.004). Low resolution melting temperture profiles derived from -<em>dF/dT </em>plots <em>in vitro </em>were similar to predictions plotted by uMELT<sup>SM</sup> <em>in silico</em>. This detection-discrimination strategy was applied to glass wool filtered water samples.</div>