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.

A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

“…16S or 16S-23S intergenic spacer region), as well as more recent efforts to detect genes such as endoglucanase , hrpB , hrpu , flic , cytochrome c 1 signal peptide, the upstream region of UDP-3- O -acyl-GlcNAc deacetylase, and a predicted glycosyl transferase [ 12 – 19 ]. For detection at the biovar 2/IIB-1&2 level, only four conventional PCR assays [ 20 – 23 ], two loop-mediated isothermal amplification (LAMP) assays [ 22 , 24 ] and two qPCR assays [ 13 , 25 ] have been developed so far.…”

“…Fegan and Prior developed the first biovar 2 specific PCR primer pair 630/631 based on Southern hybridization and competitive hybridization [ 21 ]. Although extensively used over the years, this primer set has produced false-positive reactions from strains isolated in South America [ 21 , 23 ] and targets a potential phage-related mobile element [ 20 ]. In silico genomic comparisons to identify r3b2/IIB-1&2-specific DNA for detection yielded promising results, but the specific primer pairs designed for PCR were either not fully tested [ 20 ] or targeted a potential mobile element [ 22 ].…”

“…In silico genomic comparisons to identify r3b2/IIB-1&2-specific DNA for detection yielded promising results, but the specific primer pairs designed for PCR were either not fully tested [ 20 ] or targeted a potential mobile element [ 22 ]. We recently developed a multiplex PCR assay that targets non-phage genome sequences and allows for simultaneous detection and differentiation of Rs species complex and IIB-1&2 strains with an internal plant control [ 23 ].…”

“…This set was designed based on the same, although shortened, region of the 630/631 primer pair, and was found even more susceptible to false positives [ 27 ]. A SYBR Green qPCR assay developed for r3b2/IIB-1&2 detects strain UW349, a IIB-27 strain of Rs [ 23 , 25 ]. Recently, a multiplex qPCR assay was developed to detect race 3 strains of Rs , but this assay also detects non-IIB-1&2 biovar 1 strains of Rs [ 28 ].…”

“…Imported geranium cuttings have been accidental carriers of r3b2 into the U. S. and some European countries [ 8 , 11 ], and components in extracts of this plant species strongly inhibit DNA-based amplification methods [ 23 , 25 ]. Detection of Rs in geranium cuttings requires DNA extraction methods that negate PCR inhibitors and internal reaction controls to validate the process.…”