Development of a new molecular diagnostics tool for Agrobacterium tumefaciens Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/fq977w31c

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  • Agrobacterium tumefaciens causes the economically important plant disease, crown gall. These galls are the result of abnormal growth that manifests itself on either the root or the stem and can be devastating for the ornamental plant industry. Introduction of A. tumefaciens to a nursery can be highly detrimental and thus early detection of the pathogen is crucial in order to limit its spread. Current methods of pathogen identification such as polymerase chain reaction (PCR) are costly, require specialized equipment, and can take a significant amount of time. By optimizing an isothermal recombinase polymerase amplification (RPA) and lateral flow analysis, the rapid and sensitive detection of A. tumefaciens can be done in the production setting, and does not require specialized equipment. This is possible because recombinase proteins are capable of separating double stranded DNA without the need for high temperatures. Once the RPA reaction is complete, a probe is used to bind to DNA fragments amplified from a region of the A. tumefaciens plasmid. Amplification is visualized using lateral flow analysis, aka, a dipstick, eliminating the need for gel electrophoresis. I have developed a primer set and a probe for RPA detection of phytopathogenic A. tumefaciens that is specific, sensitive, and has been optimized for reaction time, temperature, and is accurate at low DNA concentrations. I selected a gene, virD2, which is present in all known pathogenic strains of A. tumefaciens as the target for RPA. The primer set has been optimized for high specificity, yet can identify various A. tumefaciens strains. The assay was tested against phytopathogenic and non-pathogenic bacteria as well as plant samples and was shown to be specific to virD2-containing A. tumefaciens. I have also determined the ideal temperature and time for an RPA is 37℃ for 30 minutes. By optimizing these conditions, the presence or absence of A. tumefaciens can be accurately detected by applying the RPA product to the lateral flow analysis strip. If present, a band will appear on the strip in less than one minute. Development of this assay will provide growers with a fast, inexpensive, and accurate method of detecting pathogenic A. tumefaciens.
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