Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR.
OBJECTIVE
The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains.


RESULTS
A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR.


CONCLUSIONS
The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method.


CONCLUSIONS
This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.
Weller
S A
SA
Central Science Laboratory, DEFRA, York, UK. s.weller@csl.gov.uk
Stead
D E
DE
D018553:Cucumis sativus / Q000382:microbiology*; D003470:Culture Media; D005456:Fluorescent Dyes; D018551:Solanum lycopersicum / Q000382:microbiology*; D010935:Plant Diseases / Q000382:microbiology*; D018517:Plant Roots / Q000382:microbiology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012231:Rhizobium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D003470:Culture Media; D005456:Fluorescent Dyes; D019914:Taq Polymerase