Engineering a simple, rapid, inexpensive, nucleic acid based system for strain selective detection of the bacterial wilt pathogen Ralstonia solanacearum
Engineering a simple, rapid, inexpensive, nucleic acid based system for strain selective detection of the bacterial wilt pathogen Ralstonia solanacearum
Date
2012-05
Authors
Kubota, Ryo
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[Honolulu] : [University of Hawaii at Manoa], [May 2012]
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Abstract
The bacterial wilt pathogen Ralstonia solanacearum (Rs) infects a wide range of plant species. Of special concern are certain cold-tolerant strains of the bacteria designated Rs Race 3 Biovar 2 (R3B2), which collectively have been classified as a select agent in the U.S. New rapid diagnostic tools are urgently needed to discriminate these strains from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. For rapid classification of individual populations of Rs, DNA based techniques are necessary because existing immunological probes cannot resolve between populations, and classical metabolic and host typing assays require weeks of meticulous screening. Isothermal DNA amplification technologies were investigated as alternatives to PCR for pathogen identification in the field without the expensive, power-intensive equipment needed for rapid thermal cycling. Loop-mediated isothermal AMPlification (LAMP) was selected for detailed investigation based on favorable performance in preliminary work, including the ability to detect Rs in soil drainage and plant tissue. In silico whole genome bioinformatic analysis was used to identify several sequences potentially specific to R3B2, from which 3 sets of LAMP primers were designed which were highly specific to R3B2. To facilitate detection with LAMP, an innovative quenching-based probe architecture (Assimilating Probe) was developed for monitoring the LAMP reaction in real-time. The new probe is the first of its kind demonstrated for sequence-specific monitoring of the LAMP reaction, enabling rapid and reliable detection compared to non-specific intercalating dyes, eliminating false positive classifications resulting from non-specific amplification, and allowing multiplexed detection of multiple genes simultaneously and the use of internal controls within each reaction. The new real-time assay with Assimilating Probes was implemented in a custom, low-power, handheld device costing about $200. The new real time LAMP assay coupled with the new handheld devices may greatly enhance the diagnostic capabilities of investigators and may supplant the use of more expensive technologies. Given their low cost, simplicity, and portability, the new technologies may also expand the scope of gene-based testing in applications for which diagnostics were previously cost-prohibitive.
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Ph.D. University of Hawaii at Manoa 2012.
Includes bibliographical references.
Includes bibliographical references.
Keywords
Ralstonia solanacearum,
bioengineering
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering.
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