Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanace arum strains. two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (B2) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A, Amplification of t he target was measured by the 5' nuclease activity of Tag DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performe d with DNA extracted from 42 R. solanacearum and genetically or serological ly related strains to demonstrate the specificity of the assay. In pure cul tures, detection of R. solanacearum to greater than or equal to 10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed w ith inoculated potato tissue extracts, prepared by currently recommended ex traction procedures. A third fluorogenic probe (COX), designed with the pot ato cytochrome oxidase gene sequence, was also developed for use as an inte rnal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possi bility of automating the technique, offer potential advantages in routine i ndexing of potato tubers and other plant material for the presence of R. so lanacearum.