Development of an Integrated Rapid Detection System for Discrimination of Ralstonia Solanacearum Strains in the Environment

NON-TECHNICAL SUMMARY: Ralstonia solanacearum is a bacteria which causes a devastating wilting disease on a wide range of economically important crops. Annual losses due to potato affecting strains of the bacteria are estimated at above $950 million globally, though so far these strains have not been introduced to potato growing regions of North America. Rapid detection systems are essential for establishing effective quarantine of imported plant materials which may be infected with the bacteria but which may not show symptoms of the disease. In addition, rapid detection systems are required to implement effective measures to prevent disease spread once introduced to an area. The purpose of this project is to develop rapid detection systems to detect economically important pathogens such as the potato affecting strains of Ralstonia solanacearum and distinguish them from similar but less virulent strains. <P>

APPROACH: For objective 1, standard molecular techniques and bioinformatics tools will be used to isolate, identify, and characterize genetic sequences which may be used to differentiate strains of a single bacterial species (Ralstonia solanacearum) which may exhibit significant differences in virulence and economic impact to agricultural production. For objective 2, antibodies against Ralstonia solanacearum will be immobilized onto a magnetic beads. These will be used in a custom designed magnetically stabilized fluidized bed (MSFB) reactor to rapidly capture and concentrate bacteria of the target species from a sample (extracts from plant tissue, soil, or irrigation water). In addition, isothermal DNA amplification methods using strand displacing polymerases will be investigated for adaptation and compatibility with the chosen electrochemical DNA detection technology (see objective 3 below). Strand displacing polymerases continuously synthesize DNA based on circular single stranded templates, or linear DNA templates when using specially designed primers. We will be investigating alternative molecular designs for primers which will allow isothermal amplification which will allow simpler and less energy intensive temperature control, and improve the sensitivity and selectivity of detection. For objective 3, we will be developing a disposable electrode for the direct label free detection of DNA hybridization to a specific target sequence. Preliminary investigation has shown detection limits approaching 1 fM using this technology, and specificity can be achieved by controlling the hybridization conditions such as the extraction buffer composition and temperature. We intend to customize our own electrodes to provide multiple sequence and controls on the same electrode to reduce false positives and negatives, and also include a miniature integral heater to achieve temperature control for DNA amplification and selective hybridization. Finally, for objective 3 we will build a portable instrument to perform the electrochemical measurements on the disposable electrodes. For objective 4, we will test the integrated system in the lab and in cropping systems in the greenhouse to demonstrate the proof of principle of using the technology to distinguish closely related bacteria in the field.