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Location: Floral and Nursery Plants Research

Title: A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

item Stulberg, Michael - Orise Fellow
item Huang, Qi

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2015
Publication Date: 10/2/2015
Citation: Stulberg, M.J., Huang, Q. 2015. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum. PLoS One. doi: 10.1371/journal.pone.0139637.

Interpretive Summary: A bacterium causes a serious brown rot disease in potato and is not allowed to enter the United States. To make sure that plants imported from other countries do not carry this particular bacterium, a fast, accurate and reliable method to detect it is needed. We improved current methods to allow detection of the bacterium in quantity, real time and with confidence. We also simplified a way to prepare plant samples for detection of the bacterium. Our methods will benefit government officials by helping them make timely and appropriate recommendations to prevent this bacterium from getting into and spreading throughout the U.S.

Technical Abstract: Ralstonia solanacearum race 3 biovar 2 strains have the ability to cause brown rot of potato in temperate climates. Since these strains are not established in the U.S. and because of the potential risk they pose to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade. Molecular classification has grouped the race 3 biovar 2 strains into a subset of phylotype II in the R. solanacearum species complex. We designed two primer and probe sets to recognize select agent strains, a primer and probe set that recognizes the newly proposed R. solanacearum strains (current phylotype II strains), and internal plant primers and probes that can recognize eight plant species including both potato and cultivated geranium. We multiplexed one of the select agent-specific primer and probe sets with the phylotype II and plant internal control sets to provide two layers of identification of a positive select agent sample, and to validate plant extracts and also exclude false negatives. Uniplex and multiplex qPCR reactions with hydrolysis probes were tested successfully against 90 R. solanacearum species complex strains, including 34 select agent, 52 phylotype II, and nine out-group bacterial strains. The multiplex qPCR assay was also tested against 48 symptomatic samples from multiple plant hosts, and 86 asymptomatic samples from tomato and geranium. Furthermore, we designed and tested a buffer for quick and easy DNA extraction from geranium tissue for use in qPCR assays.