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Detection of Ralstonia solanacearum by Loop-Mediated Isothermal Amplification

September 2008 , Volume 98 , Number  9
Pages  1,045 - 1,051

R. Kubota, B. G. Vine, A. M. Alvarez, and D. M. Jenkins

First and fourth authors: Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu 96822; and second and third authors: Department of Plant and Environmental Protection Sciences, University of Hawaii, Honolulu 96822.

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Accepted for publication 22 May 2008.

Ralstonia solanacearum is a pathogenic bacterium that causes wilt in over 200 plant species. Here we report a rapid and sensitive detection of R. solanacearum using an isothermal method for copying DNA known as loop-mediated amplification (LAMP). A set of four primers was designed to replicate the gene coding for the flagellar subunit, fliC, and conditions for detection were optimized to complete in 60 min at 65°C. Magnesium pyrophosphate resulting from the amplification reaction could be detected optically as an increase in the solution turbidity, and the DNA products spread in a reproducible ladder-like banding pattern after electrophoresis in an agarose gel. Replication of the fliC gene was detected only from R. solanacearum. The detection limit of this LAMP assay was between 104 to 106 colony forming units/ml, and the technique may be useful for developing rapid and sensitive detection methods for the R. solanacearum pathogen in soil and water.

Additional keywords:gene-based diagnostics.

© 2008 The American Phytopathological Society