title: Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato.
abstract: Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least 1.47×10(2) copies/μl of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or 10(-6) dilution point of 0.12 at OD600 units of cells per reaction using a calibrated cell suspension.
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-550, Korea ; Department of Microbiology, Dankook University, Cheonan 330-714, Korea.
authors.forename: Min Seok
authors.initials: MS
authors.lastname: Cho
authors.affiliation: Department of Applied Biology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon 200-701, Korea.
authors.forename: Duck Hwan
authors.initials: DH
authors.lastname: Park
authors.affiliation: Department of Applied Biology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon 200-701, Korea.
authors.forename: Min
authors.initials: M
authors.lastname: Namgung
authors.affiliation: Department of Microbiology, Dankook University, Cheonan 330-714, Korea.
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Ahn
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-550, Korea.
authors.forename: Dong Suk
authors.initials: DS
authors.lastname: Park
keywords: Clavibacter michiganensis subsp. sepedonicus; bacterial ring rot; detection; potato; putative phage-related protein; real-time PCR