SENSITIVE AND SPECIFIC DETECTION OF XANTHOMONAS-CAMPESTRIS PV PELARGONII WITH DNA PRIMERS AND PROBES IDENTIFIED BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS
S. Manulis et al., SENSITIVE AND SPECIFIC DETECTION OF XANTHOMONAS-CAMPESTRIS PV PELARGONII WITH DNA PRIMERS AND PROBES IDENTIFIED BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS, Applied and environmental microbiology, 60(11), 1994, pp. 4094-4099
The random amplified polymorphic DNA method was used to distinguish st
rains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomon
as species and/or pathovars. Among the 42 arbitrarily chosen primers e
valuated, 3 were found to reveal diagnostic polymorphisms when purifie
d DNAs from compared strains were amplified by the PCR. The three prim
ers revealed DNA amplification patterns which were conserved among all
53 strains tested of X. campestris pv. pelargonii isolated from vario
us locations worldwide. The distinctive X. campestris pv. pelargonii p
atterns were clearly different from those obtained with any of 46 othe
r Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, appare
ntly unique to X. campestris pv. pelargonii by these random amplified
polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA pr
obe. It hybridized with total DNA from all 53 X. campestris pv. pelarg
onii strains tested and not with any of the 46 other Xanthomonas strai
ns tested. The DNA sequence of the terminal ends of this 1.2-kb fragme
nt was obtained and used to design a pair of 18-mer oligonucleotide pr
imers specific for X. campestris pv. pelargonii. The custom-synthesize
d primers amplified the same 1.2 kb DNA fragment from all 53 X. campes
tris pv. pelargonii strains tested and failed to amplify DNA from any
of the 46 other Xanthomonas strains tested. DNA isolated from saprophy
tes associated with the geranium plant also did not produce amplified
DNA with these primers. The sensitivity of the PCR assay using the cus
tom-synthesized primers was between 10 and 50 cells. The techniques us
ed could provide a general method to identify pathovar-specific DNA pr
imers for rapid, sensitive, and specific detection and identification
of plant-pathogenic bacteria.