Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

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Abstract
Aims: The development of a fluorogenic, 5′ nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. Methods and 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. Significance and Impact of the Study: 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.