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Loop-mediated isothermal amplification for detection phytopathogenic bacteria
STEHLÍKOVÁ, Dagmar
In the areas of tomato and pepper growth, Xanthomonas related bacteria cause high economical losses every year. This thesis presents development of three separate LAMP assays for portable rapid and sensitive detection of plant pathogenic bacteria X. vesicatoria, X. gardneri and X. euvesicatoria that cause bacterial spot of tomato and pepper. Based on bioinformatic analysis of available gene sequences was chosen one gene efp for design primers for X. vesicatoria. The reaction was optimized by mixing the components where MgSO4 was in a gradient dilution. The LAMP method for detection X. vesicatoria was transferred to a Smart-DART platform (Diagenetix, USA). The atpD gene was selected to design primers for specific detection of X. gardneri. Real-time LAMP and colorimetric LAMP were optimized. LAMP method was optimized for X. euvesicatoria. It was based on the selection of a specific region within the whole genome. The designed primers were tested at two different concentrations. The reaction was specific at both concentrations. The difference was only at the beginning of the threshold fluorescence overhang. To verify the functionality of the LAMP method, the method was tested on DNA isolated directly from plants. The LAMP for specific detection of X. euvesicatoria was detection limit of 0,128 pg/ěl DNA in the reaction. Fusarium circinatum is the causal agent of pitch canker, a destructive disease, which is an economically important disease of pines and other conifers in many parts of the world including south North America, Central and South America, east Asia, South Africa and also south-west Europe. It causes cankers from which a huge amount of resin is extruded, especially at the early stage of colonization. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications. For this reason, in this thesis is developed a sequence-specific probe LAMP assay for F. circinatum using a field-deployable portable Genie?II (Optigene, UK) and Smart-DART? (Diagenetix, USA) instrument. The assay is able to detect target DNA in pine tissue in a reasonably short time and detection limit was as low as 0,05 pg ěL.1 of DNA. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains collected in different regions of the iv world and host plants, giving reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.
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