A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum

Authors: Stulberg, Michael; Shao, Jonathan; Huang, Qi

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/12/2014
Publication Date: 9/22/2014
Citation: Stulberg, M.J., Shao, J.Y., Huang, Q. 2014. A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum. Applied and Environmental Microbiology. 99:333-341.

Interpretive Summary: Ralstonia solanacearum causes bacterial wilt, a soil-borne vascular disease that is distributed worldwide and attacks over 450 plant species including ornamentals such as geranium. It also limits the production of such economically important crops as tomato, tobacco, potato and banana. R. solanacearum is generally classified into 5 races based on host range and 5 biovars based on biochemical properties. R. solanacearum race 3, biovar 2 (r3b2) causes destructive brown rot of potato, and is a quarantined and select agent pathogen in the U. S., subject to the strictest quarantine and eradication regulations. The current DNA-based detection tool for R. solanacearum r3b2 targets a potential mobile DNA sequence and false positives or false negatives have been reported. In addition, no assay is currently available to allow detection of R. solanacearum at both the species and r3b2 levels in a single reaction. Such an assay is urgently needed in light of a recent congressional mandate to define all R. solanacearum strains as Select Agent until further testing can exclude them from r3b2 strains. Therefore, the goal of our research was to find a stable DNA region for testing and to combine the detection assays at the species and r3b2 levels into one protocol to save on cost and response time. We successfully identified new regions of the R. solanacearum genome that are more stable than previously targeted regions (i.e. non-mobile). Additionally, we found a sequence that identifies the R. solanacearum species and have combined that with our detection of the R. solanacearum r3b2 individual. We also included a positive control in the assay to verify that the test was correctly performed. Our improved assay will decrease false positives by targeting non-mobile DNA, and increase the efficiency of identifying both an R. solanacearum positive sample and, specifically, whether it is the strain we are most concerned about not introducing into the U.S. The assay will benefit diagnosticians, county agents, and state and government officials by helping them make timely and appropriate decisions to prevent and exclude r3b2 from becoming established in the U.S.

Technical Abstract: Ralstonia solanacearum causes bacterial wilt in a variety of cash crops. R. solanacearum race 3 biovar 2 strains are considered select agents by the U.S. Government because they are not endemic to the U.S. and have the potential to cause brown rot disease in our potato production fields. Simple and accurate methods are needed for quick identification prior to more characteristic but time-consuming verification methods. We developed a multiplex PCR assay that identifies the R. solanacearum species complex strain, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions all in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and also for select agent strains, using in silico genome subtraction. We also designed and included internal plant DNA control primers. We tested the multiplex against 90 R. solanacearum species complex strains, 34 of them select agents, and five out-group bacterial species. As expected, a species-complex specific band of 296-bp was amplified from all the 90 R. solanacearum species complex strains, and a select agent specific band of 132-bp was amplified from only the 34 select agents. Additionally, the multiplex successfully detected plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco. Rapid, accurate, and reliable detection and identification of select agent pathogens is critical for diagnosticians, county agents, and state and government officials, in order to make timely and appropriate recommendations to exclude the bacterium from the United States.