Use of molecular beacons for direct detection of Loop-mediated isothermal AMPlification (LAMP) amplicons of the plant pathogen Ralstonia solanacearum.
R. KUBOTA (1), G. D. Peckham (1), A. M. Alvarez (1), D. M. Jenkins (1) (1) University of Hawaii at Manoa, Honolulu, HI, USA.
Phytopathology 99:S68.

Abstract: Isothermal DNA amplification techniques, such as Loop-mediated isothermal AMPlification (LAMP), are suitable for rapid detection of bacteria with simple field devices due to the ability to amplify DNA with high specificity, efficiency, and speed without thermal cycling. However, an additional step to visualize the product helps confirm the presence of the amplicons and ensure specificity of the reaction. Molecular beacons were designed to detect specific LAMP amplicons that distinguished several unique populations of the bacterial wilt pathogen Ralstonia solanacearum (Rs). Sequence-specific molecular beacons were designed to target loop regions, which are unique single-stranded DNA sequences generated by the LAMP primer sets. The reaction of the molecular beacon with the loop region distinguished group-specific amplicons generated by LAMP primer sets from a variety of closely related Rs strains. Group-specific LAMP was evaluated against 274 strains of Rs representing three phylotypes, four races and four biovars. Rs-specific LAMP products were detected in samples containing target strains but not in samples containing other Rs strains or non-target species. Early prototypes suggest that the LAMP reaction coupled with DNA hybridization probes can form the basis of a simple rapid diagnostic system for subgroups of R. solanacearum.