Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato

• Main Authors: Min Seok Cho, Duck Hwan Park, Min Namgung, Tae-Young Ahn, Dong Suk Park
• Format: Article
• Language: English
• Published: Hanrimwon Publishing Company 2015-06-01
• Series: The Plant Pathology Journal
• Subjects: bacterial ring rot; Clavibacter michiganensis subsp. sepedonicus; detection; putative phage-related protein; potato; real-time PCR
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/4453993

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 1.47×10² 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.