TITLE The design of specific primers for the detection of Ralstonia solanacearum in soil samples by polymerase chain reaction
AUTHOR Yung-An Lee
Department of Biology, Fu Jen Catholic University, Hsin Chuang 24205, Taipei, Taiwan, Republic of China
Chi-Chung Wang
Department of Biology, Fu Jen Catholic University, Hsin Chuang 24205, Taipei, Taiwan, Republic of China
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ABSTRACT A 0.7-kb DNA fragment, amplified by the randomly amplified polymorphic DNA (RAPD) method from Ralstonia solanacearum total DNA, was cloned and evaluated as a specific DNA probe. This 0.7-kb DNA fragment hybridized to a 2.7-kb EcoRI fragment in the EcoRI-digested total DNA of R. solanacearum. The 2.7-kb EcoRI fragment was also cloned and hybridized only to R. solanacearum but not to other Pseudomonas spp., pathovars of Xanthomonas campestris, and Erwinia spp. tested. The DNA sequence of this 2.7-kb fragment was obtained and used to design specific oligonucleotide primers for polymerase chain reaction (PCR) amplification. The primers amplified the same 1.1-kb PCR product from all R. solanacearum strains tested and failed to amplify DNA from any other plant pathogenic bacterial strains tested. DNA isolated from several saprophytic bacteria did not produce any PCR products with these primers. This specific PCR for R. solanacearum was also performed from colonies grown on BG medium with similar results. The sensitivity of the PCR assay using the specific primers was about 20 cells. The PCR assay was used to detect R. solanacearum in soil using these primer sets. No PCR product could be found when soil extract containing R. solanacearum was used directly in the assay. DNA extraction from soil was needed for the success of PCR assay. A simple method for DNA extraction from soil for PCR assay was developed and can hasten the detection of R. solanacearum in soil.
KEYWORD Erwinia; Polymerase chain reaction; Pseudomonas; Ralstonia solanacearum; Xanthomonas;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 41 Number 2 April 2000, page 121-128, 8 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China