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Quantitative Detection of Clavibacter michiganensis subsp. sepedonicus by Competitive Polymerase Chain Reaction. X. Hu, Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins 80523; F.-M. Lai(2), A. S. N. Reddy(3), and C. A. Ishimaru(4). (2)(4)Plant Pathology and Weed Science, Colorado State University, Fort Collins 80523; (3)Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins 80523. Phytopathology 85:1468-1473. Accepted for publication 20 September 1995. Copyright 1995 The American Phytopathological Society. DOI: 10.1094/Phyto-85-1468.

A competitive polymerase chain reaction (PCR) method was developed for detection and quantification of Clavibacter michiganensis subsp. sepedonicus DNA in infected plant tissues. An internal standard DNA template that served as a control for all PCR tests was generated by amplification of Arabidopsis genomic DNA under low annealing temperatures with primers specific for C. michiganensis subsp. sepedonicus. The 450-bp product amplified from the internal standard DNA template was distinct from the 250-bp product characteristic of C. michiganensis subsp. sepedonicus. The ratio of the PCR products amplified in the presence of a constant amount of internal standard DNA template increased linearly and competitively with increased amounts of C. michiganensis subsp. sepedonicus DNA. Cell numbers estimated by immuno-fluorescence antibody staining (IFAS) were consistent with PCR product ratios obtained from cell cultures and inoculated potato plantlets. Compared to IFAS, competitive PCR was about 10-fold more sensitive and detected as few as 100 cells. Competitive PCR should greatly reduce misdiagnosis from false negatives and provide a quantitative approach for estimating pathogen population sizes in samples.