Quantitative Detection of<i>Clavibacter michiganensis</i>subsp.<i>sepedonicus</i>by Competitive Polymerase Chain Reaction

Abstract

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 immunofluorescence 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.