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 solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal 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 possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.
Weller
S A
SA
Central Science Laboratory, MAFF, Sand Hutton, York, YO41 1LZ, United Kingdom. s.weller@csl.gov.uk
Elphinstone
J G
JG
Smith
N C
NC
Boonham
N
N
Stead
D E
DE
D020562:Betaproteobacteria / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015342:DNA Probes; D005456:Fluorescent Dyes; D010935:Plant Diseases / Q000382:microbiology; D010936:Plant Extracts / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000382:microbiology; D019914:Taq Polymerase / Q000378:metabolism*
D015342:DNA Probes; D005456:Fluorescent Dyes; D010936:Plant Extracts; D019914:Taq Polymerase