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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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Evaluation of rheological properties of terbinafine hydrochloride creams
Stehlíková, Dagmar ; Šnejdrová, Eva (advisor) ; Holas, Ondřej (referee)
CHARLES UNIVERSITY Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Mgr. Dagmar Stehlíková Consultant: PharmDr. Eva Šnejdrová, Ph.D. Title of Thesis: Evaluation of rheological properties of terbinafine hydrochloride creams The aim of the rigorous work was to evaluate the rheological properties of creams with terbinafine hydrochloride at different stages of the formulation of a generic preparation using a suitable methodology and their comparison with the commercial preparation Lamisil 10 mg/g crm. (GlaxoSmithKline), which was specified as a standard. The theoretical part deals with the characterization of generic drugs, pharmaceutical and therapeutic equivalence. Tests suitable for measuring the flow properties of semi-solid preparations and evaluation methods are presented. The properties of terbinafine hydrochloride and the process of developing generic semi-solid formulations are summarized. Testing was performed at 25 řC on a Kinexus Pro+ rotational rheometer using cone-plate geometry. The flow properties of the creams were evaluated by nonequilibrium flow and viscosity curves, which were analysed by the power law model and Herschel-Bulkely model. The coefficient of consistency, the index of flow behaviour and the yield point were expressed. The...
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Design and testing of multiplex-PCR primers for detection of bacterial spot of tomato
STEHLÍKOVÁ, Dagmar
The subject of this work is to develop multiplex-PCR assay for specific detection of plant pathogenic bacteria of Xanthomonas genus causing bacterial spot of tomato. PCR primers for detection of groups A (X. euvesicatoria), B (X. vesicatoria), C (X. perforans) and D (X. gardneri) were developed based on the DNA sequences obtained by sequencing and from the GenBank database (NCBI). Four primer pairs - Xe_shotgun_104, Xe_shotgun_1819, Xv_atpD_403, Xp_efP_202 were designed and subsequently thoroughly tested and optimized for parallel detection of these bacteria. Specificity of the primers was tested on a large complex of bacterial strains pathogenic to tomato and related crops. Following the protocol described above X. vesicatoria, X. euvesicatoria, X. perforans and X. gardneri can be quickly and reliably identified in a single multiplex-PCR assay.
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Genotyping of bacterial genus \kur{Xanthomonas} pathogenic for tomato and pepper by multilocus sequencing.
STEHLÍKOVÁ, Dagmar
The subject of this bachelor thesis is to estimate the degree of relationship of 20 collected isolated samples of Xanthomonas genus causing bacterial spot of tomatoes and peppers. To assess the relationship, the method of multilocus sequence analysis has been used (MLSA). Sequences of genes atpD, rpoD, gyrB, glnA and efP were found and analyzed using MEGA software version 5.1. The result was evaluated using a dendrogram according to which the isolates were divided into two clusters. The first cluster includes isolated of Xanthomonas euvesicatoria belonging to group A. The second cluster includes isolates of the species Xanthomonas vesicatoria belonging to group B. The results obtained in this work are consistent with the present classification of the xanhtomonads causing bacterial spot of tomatoes and peppers.
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