Application of loop-mediated isothermal amplification method (LAMP) for detection of the bacterial wilt pathogen Ralstonia solanacearum in environmental samples. R. KUBOTA (1), M. L. Paret (1), A. M. Alvarez (1), D. M. Jenkins (1)
(1) University of Hawaii at Manoa, Honolulu, HI, USA
Phytopathology 98:S85

Abstract: DNA-based detection methods can provide improved specificity and sensitivity compared to serological methods. However, these methods remain impractical for most field applications due to difficulties in concentrating the organism from the environmental samples and ensuring that enough DNA is available for detection. Isothermal DNA amplification techniques, such as Loop-mediated isothermal AMPlification (LAMP), might be suitable for rapid field detection because of its ability to amplify DNA with high specificity, efficiency, and speed without thermal cyclers. The objective of this research was to develop a rapid detection method for Ralstonia solanacearum (Rs) in environmental samples. To demonstrate the application of LAMP in environmental samples, edible ginger plants (Zingiber officinale) were infected with Rs, and effluent water samples were collected from daily irrigation water. A simple filtration technique was applied to concentrate bacteria, followed by a species-specific LAMP and pathogen presence was visually determined as the precipitation of an insoluble pyrophosphate by-product of amplification, which increased the turbidity of the solution. The detection limit of the direct LAMP assay was between 10(^3) – 10(^5) CFU/ml, which is the same as PCR and more sensitive than ELISA. LAMP shows promise for not only rapid detection but also for discrimination of closely related sub population of Rs in soil and water.