A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum

Abstract

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.